The Mycota

Edited by
K. Esser
The Mycota

I Growth, Differentiation and Sexuality

1st edition ed. by J.G.H. Wessels and F. Meinhardt
2nd edition ed. by U. Kües and R. Fischer

II Genetics and Biotechnology

Ed. by U. Kück

III Biochemistry and Molecular Biology

Ed. by R. Brambl and G. Marzluf

IV Environmental and Microbial Relationships

1st edition ed. by D. Wicklow and B. Söderström
2nd edition ed. by C.P. Kubicek and I.S. Druzhinina

V Plant Relationships
1st edition ed. by G. Carroll and P. Tudzynski
2nd edition ed. by H.B. Deising

VI Human and Animal Relationships

1st edition ed. by D.H. Howard and J.D. Miller
2nd edition ed. by A.A. Brakhage and P.F. Zipfel

VII Systematics and Evolution

Ed. by D.J. McLaughlin, E.G. McLaughlin, and P.A. Lemke†

VIII Biology of the Fungal Cell

Ed. by R.J. Howard and N.A.R. Gow

IX Fungal Associations
Ed. by B. Hock

X Industrial Applications
1st edition ed. by H.D. Osiewacz
2nd edition ed. by M. Hofrichter and R. Ullrich

XI Agricultural Applications
Ed. by F. Kempken

XII Human Fungal Pathogens

Ed. by J.E. Domer and G.S. Kobayashi

XIII Fungal Genomics

Ed. by A.J.P. Brown

XIV Evolution of Fungi and Fungal-like Organisms

Ed. by J. Wöstemeyer

XV Physiology and Genetics: Selected Basic and Applied Aspects

Ed. by T. Anke and D. Weber
The Mycota
A Comprehensive Treatise
on Fungi as Experimental Systems
for Basic and Applied Research

Edited by K. Esser

XV Physiology and Genetics

Selected Basic and
Applied Aspects

Volume Editors:
T. Anke and D. Weber
Series Editor
Professor Dr. Dr. h.c. mult. Karl Esser
Allgemeine Botanik
Ruhr-Universität
44780 Bochum, Germany
Tel.: +49 (234)32-22211
Fax.: +49 (234)32-14211
e-mail: Karl.Esser@rub.de

Volume Editors

Professor Dr. Timm Anke

Institute for Biotechnology and Drug Research,
IBWF e.V., Erwin-Schroedinger-Strasse 56,
67663 Kaiserslautern, Germany
Tel.: +49 (631) 205-2697
Fax: +49 (631) 205-2699
e-mail: timm.anke@ibwf.de

Dr. Daniela Weber

MIP International Pharma Research GmbH
Mühlstr. 50
66386 St. Ingbert, Germany
Tel.: +49 (163) 3111278
e-mail: weber-daniela@gmx.de

Library of Congress Control Number: 2009926066

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Karl Esser
(born 1924) is retired Professor of General Botany and Director
of the Botanical Garden at the Ruhr-Universität Bochum
(Germany). His scientific work focused on basic research in
classical and molecular genetics in relation to practical
application. His studies were carried out mostly on fungi.
Together with his collaborators he was the first to detect
plasmids in higher fungi. This has led to the integration of
fungal genetics in biotechnology. His scientific work was
distinguished by many national and international honors,
especially three honorary doctoral degrees.

Timm Anke
(born 1944) studied Biochemistry at the University of Tuebin-
gen, where he completed his PhD degree with a dissertation on
the biosynthesis of fungal siderophores. In 1973 he joined Fritz
Lipmann’s group of at the Rockefeller University in New York
City, where he investigated the biosynthesis of valinomycin, a
streptomycete ionophore. After his return to Tuebingen in 1975
he started organizing a group searching for new antibiotics from
basidiomycetes within the framework of Hans Zaehner’s
Collaboratorive Research Center (SFB 76) focusing on the
chemistry and biology of microorganisms. In 1981 he became
full Professor of Biotechnology at the University of Kaiserslau-
tern and since 1998 he has headed the Institute of Biotechnology
and Drug Research (IBWF e. V.) in Kaiserslautern. One of his
outstanding achievements in the field of antibiotic research is
the discovery of the strobilurins, a major class of agricultural
fungicides, for which he was awarded the Karl-Heinz-Beckurts
Prize in 1996.

Daniela Weber
(born 1978) studied natural products from endophytic fungi
during her graduate work at the University of Kaiserslautern’s
Department of Biotechnology. She received a Chemiefonds
fellowship for Ph.D. students. Her Ph.D., completed in 2006,
focused on the isolation of endophytic fungi from medicinal
plants and the investigation of the fungal metabolites. She later
continued her work at the Institute of Biotechnology and Drug
Research (IBWF, Kasiserslautern). In 2008 she accepted a
position at MIP International Pharma Research GmbH (St.
Ingbert, Germany). Her fields of activities are pharmaco-
toxicological and clinical expert reports, pharmacovigilance,
and regulatory affairs.
Series Preface

Mycology, the study of fungi, originated as a sub discipline of botany and was a
descriptive discipline, largely neglected as an experimental science until the early
years of this century. A seminal paper by Blakeslee in 1904 provided evidence for self
incompatibility, termed “heterothallism”, and stimulated interest in studies related to
the control of sexual reproduction in fungi by mating-type specificities. Soon to follow
was the demonstration that sexually reproducing fungi exhibit Mendelian inheritance
and that it was possible to conduct formal genetic analysis with fungi. The names
Burgeff, Kniep and Lindegren are all associated with this early period of fungal
genetics research.
These studies and the discovery of penicillin by Fleming, who shared a Nobel Prize
in 1945, provided further impetus for experimental research with fungi. Thus began a
period of interest in mutation induction and analysis of mutants for biochemical
traits. Such fundamental research, conducted largely with Neurospora crassa, led to
the one gene: one enzyme hypothesis and to a second Nobel Prize for fungal research
awarded to Beadle and Tatum in 1958. Fundamental research in biochemical genetics
was extended to other fungi, especially to Saccharomyces cerevisiae, and by the mid-
1960s fungal systems were much favored for studies in eukaryotic molecular biology
and were soon able to compete with bacterial systems in the molecular arena.
The experimental achievements in research on the genetics and molecular biology of
fungi have benefited more generally studies in the related fields of fungal biochemistry,
plant pathology, medical mycology, and systematics. Today, there is much interest in
the genetic manipulation of fungi for applied research. This current interest in
biotechnical genetics has been augmented by the development of DNA-mediated
transformation systems in fungi and by an understanding of gene expression and
regulation at the molecular level. Applied research initiatives involving fungi extend
broadly to areas of interest not only to industry but to agricultural and environmental
sciences as well.
It is this burgeoning interest in fungi as experimental systems for applied as well as
basic research that has prompted publication of this series of books under the title
The Mycota. This title knowingly relegates fungi into a separate realm, distinct from
that of either plants, animals, or protozoa. For consistency throughout this Series of
Volumes the names adopted for major groups of fungi (representative genera in
parentheses) areas follows:
Pseudomycota
Division: Oomycota (Achlya, Phytophthora, Pythium)
Division: Hyphochytriomycota
Eumycota
Division: Chytridiomycota (Allomyces)
Division: Zygomycota (Mucor, Phycomyces, Blakeslea)
viii Series Preface

Division: Dikaryomycota
Subdivision: Ascomycotina
Class: Saccharomycetes (Saccharomyces, Schizosaccharomyces)
Class: Ascomycetes (Neurospora, Podospora, Aspergillus)
Subdivision: Basidiomycotina
Class: Heterobasidiomycetes (Ustilago, Tremella)
Class: Homobasidiomycetes (Schizophyllum, Coprinus)
We have made the decision to exclude from The Mycota the slime molds which,
although they have traditional and strong ties to mycology, truly represent nonfungal
forms insofar as they ingest nutrients by phagocytosis, lack a cell wall during the
assimilative phase, and clearly show affinities with certain protozoan taxa.
The Series throughout will address three basic questions: what are the fungi, what
dothey do, and what is their relevance to human affairs? Such a focused and
comprehensive treatment of the fungi is long overdue in the opinion of the editors.
A volume devoted to systematics would ordinarily have been the first to appear in
this Series. However, the scope of such a volume, coupled with the need to give
serious and sustained consideration to any reclassification of major fungal groups,
has delayed early publication. We wish, however, to provide a preamble on the nature
off ungi, to acquaint readers who are unfamiliar with fungi with certain characteristics
that are representative of these organisms and which make them attractive subjects
for experimentation.
The fungi represent a heterogeneous assemblage of eukaryotic microorganisms.
Fungal metabolism is characteristically heterotrophic or assimilative for organic
carbon and some nonelemental source of nitrogen. Fungal cells characteristically
imbibe or absorb, rather thaningest, nutrients and they have rigid cellwalls. The vast
majority of fungi are haploid organisms reproducing either sexually or asexually
through spores. The spore forms and details on their method of production have been
used to delineate most fungal taxa. Although there is amultitude of spore forms,
fungal spores are basically only of two types: (i) asexual spores are formed following
mitosis (mitospores) and culminate vegetative growth, and (ii) sexual spores are
formed following meiosis (meiospores) and are borne in or upon specialized
generative structures, the latter frequently clustered in a fruit body. The vegetative
forms of fungi are either unicellular, yeasts are an example, or hyphal; the latter may
be branched to form an extensive mycelium.
Regardless of these details, it is the accessibility of spores, especially the direct
recovery of meiospores coupled with extended vegetative haploidy, that have made
fungi especially attractive as objects for experimental research.
The ability of fungi, especially the saprobic fungi, to absorb and grow on rather
simple and defined substrates and to convert these substances, not only into essential
metabolites but into important secondary metabolites, is also noteworthy. The
metabolic capacities of fungi have attracted much interest in natural products
chemistry and in the production of antibiotics and other bioactive compounds. Fungi,
especially yeasts, are important in fermentation processes. Other fungi are important
in the production of enzymes, citric acid and other organic compounds as well as in
the fermentation of foods.
Fungi have invaded every conceivable ecological niche. Saprobic forms abound,
especially in the decay of organic debris. Pathogenic forms exist with both plant and
animal hosts. Fungi even grow on other fungi. They are found in aquatic as well as soil
environments, and their spores may pollute the air. Some are edible; others are
 ix

poisonous. Many are variously associated with plants as copartners in the formation
of lichens and mycorrhizae, as symbiotic endophytes or as overt pathogens.
Association with animal systems varies; examples include the predaceous fungi that
trap nematodes, the micro fungi that grow in the anaerobic environment of the
rumen, the many insect associated fungi and the medically important pathogens
afflicting humans. Yes, fungi are ubiquitous and important.
There are many fungi, conservative estimates are in the order of 100,000 species,
and there are many ways to study them, from descriptive accounts of organisms
found in nature to laboratory experimentation at the cellular and molecular level. All
such studies expand our knowledge of fungi and of fungal processes and improve our
ability to utilize and to control fungi for the benefit of humankind.
We have invited leading research specialists in the field of mycology to contributeto
this Series. We are especially indebted and grateful for the initiative and leadership
shown by the Volume Editors in selecting topics and assembling the experts. We have
all been a bit ambitious in producing these Volumes on a timely basis and there in lies
the possibility of mistakes and oversights in this first edition. We encourage the
readership to draw our attention to any error, omission or inconsistency in this Series
in order that improvements can be made in any subsequent edition.
Finally, we wish to acknowledge the willingness of Springer-Verlag to host this
project, which is envisioned to require more than 5 years of effort and the publication
of at least nine Volumes.

Bochum, Germany KARL ESSER

Auburn, AL, USA PAUL A. LEMKE
April 1994 Series Editors
It is Time to Retire

During the Fourth International Mycological Congress in Regensburg (1989) while

relaxing in a beer garden with Paul Lemke (USA), Dr. Czeschlik (Springer Verlag)
discussed with us the possibility to publish a series about Fungi. We both were at first
somewhat reserved, but after a comprehensive discussion this idea looked promising.
In analogy to another series by Springer Verlag entitled The Prokaryota we decided
to name the new series The Mycota.
Then Paul Lemke and I created a program involving seven volumes covering a wide
area of Mycology. The first volume was presented in 1994 at the Fifth International
Mycological Congress in Vancover (Canada). The other volumes followed step by
step. After the early death of Paul Lemke (1995) I proceeded alone as Series Editor.
Since evidently the series was well accepted by the scientific community and since the
broad area of Fungi was not completely covered, it was decided to proceed with eight
more volumes. In addition, in the following years second editions of eight volumes
were published.
Now we present Volume XV. This will be the last volume of this series. As its title
“Physiology and Genetics: Selected Basic and Applied Aspects” expresses, it contains
special papers of various fields of Mycology which have been missing in the
previous volumes.
Now, after 20 years of editing this series and at the age of 85 years, I guess it is the
right time to terminate my editorship. I would like to express my sincerest thanks to
all the volume editors, to the numerous authors for their successful cooperation and
last but not least to Joan Bennett who supported me in editing three volumes.
I would also like to thank Springer Verlag, represented by Drs. Czeschlick and
Schlitzberger for their support and cooperation.
I hope that The Mycota will also in the future find the interest of mycologists and
other scholars interrested in Fungi and maybe some more second editions.

Bochum, Germany KARL ESSER

May 2009
Volume Preface

More than 120 000 different fungal species have been described and it is estimated
that there exist more than 1.5106 species. Fungi have adopted many different ways of
living in very diverse habitats as saprophytes, pathogens, symbionts or endophytes.
Fungi and their products are used for the fermentation and processing of food and
feeds, for biological control and for the production of vitamins and amino acids. Some
of their secondary metabolites are used in medicine, e.g. as antibiotics, immuno-
supressants, cholesterol-lowering drugs or as agrochemical fungicides. Recently,
progress in the field of mycology has been substantial due to new methodological
approaches and technologies, many of them DNA-based, strongly adding to the
motivation to compile a new volume of “The Mycota”.
Mycota XV “Physiology and Genetics: Selected Basic and Applied Aspects”
provides a selection of state of the art reviews in traditional and new fields of
mycology. As addressed in Chap. 1, DNA sequencing of functionally conserved
homologue genes has resulted in new approaches to taxonomy, which hopefully will
result in phylogenetic trees and taxonomic groups which can be understood and
accepted by the majority of mycologists. As a homothallic fungus Sordaria
macrospora, producing only sexual meiospores and no asexual conidia, is an ideal
model for studying all stages of fruiting body formation and development. The
application of several genetic tools allows advanced studies of genetic interactions
controlling developmental plasticity (Chap. 2). Chapter 3 summarizes the current
knowledge of inteins, internal protein sequences, which are “selfish elements” in
fungal genomes. They are transcribed and translated together with their host
protein and excised at the protein level. Chapter 4 gives an overview of fungal
apoptosis in yeasts and filamentous fungi. Similarities and differences between
fungal and mammalian apoptosis are discussed and the role of apoptosis in
development and ageing are described and evaluated. A broad view on ways and
means of the vegetative and sexual interaction of fungal colonies as well as the
communication of fungi with bacteria, plants and animals is offered in Chap. 5. In
the interaction of yeasts killer toxins play an important role. Their structures,
modes of action and resistance as well as possible applications are discussed in
Chap. 6. Chapter 7 deals with aspects of evolutionary and ecological interactions of
fungi and insects; and Chap. 8 offers an insight into the occurrence and metabolites
of endophytic fungi. Not all compounds isolated from plants are genuine plant
metabolites but are produced by fungi. Thus the ergoline alkaloids present in
Convolvulaceae are produced by fungi living in close association with secretory
glands on the leaf surface (Chap. 9). Basidiomycetes are a rich source of unique
secondary metabolites in most cases not found in other fungi. Chapter 10 offers a
survey of new compounds isolated within the past decade, with special emphasis on
bioactive metabolites. Genome-wide approaches to identify genes or gene products
essential for the establishment of pathogenic interactions between plant host and
xiv Volume Preface

fungal pathogen are discussed in Chap. 11. In addition, the authors stress the
important role of small molecules in identifying and validating new targets for
fungicides. Helminths can pose serious problems to animal and human health. It is
therefore quite remarkable that fungi produce low molecular weight compounds
specifically interfering with reactions not present in the mammalian hosts, paving
the way to non-toxic medications or agrochemicals (Chap. 12). Chapter 13 describes
the occurrence, structures and biological activities of peptides and depsipeptides
produced by fungi and discusses the importance of these compounds as lead
compounds for agricultural and pharmaceutical applications. The sequencing of
whole fungal genomes has revealed that there are many more genes supposedly
coding for secondary metabolites than there are compounds already isolated and
characterized. As can be seen in Chap. 14, homologue overexpression of a
regulatory gene construct can indeed lead to silent gene expression and production
of the corresponding metabolite. Chapters 15–17 deal with genes, enzymes and
products of important biogenetic pathways. Nonribosomal peptide synthetases of
fungi differ in some major aspects from the corresponding bacterial enzymes. The
same is true for fungal polyketides which constitute a large part of the fungal
secondary metabolome. The importance of the detrimental mycotoxin ochratoxin
A for human health and the alimentary industry was recognized only recently. The
investigation of its biosynthesis and regulation is important for developing
strategies for its avoidance in food and feed. Chapter 18 is devoted to genetic
and metabolic engineering in fungi, key areas of research aiming at the improved
application of these organisms in biotechnology.
We do hope that readers enjoy reading this volume of The Mycota. We are very
grateful to the contributing authors, whose expertise and efforts have made this
project possible. We thank Dr. Andrea Schlitzberger of Springer Verlag for her
support and engagement during the preparation of this volume.

Kaiserslautern and St. Ingbert, Germany TIMM ANKE

May 2009 DANIELA WEBER
Volume Editors
Contents

1 Recent Developments in the Molecular Taxonomy of Fungi . . . . . . . . . . . . . . . . . . 1

ROLAND W.S. WEBER

2 Sordaria macrospora, a Model System for Fungal Development . . . . . . . . . . . . . 17

ULRICH KÜCK, STEFANIE PÖGGELER, MINOU NOWROUSIAN, NICOLE NOLTING, INES ENGH

3 Inteins – Selfish Elements in Fungal Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

SKANDER ELLEUCHE, STEFANIE PÖGGELER

4 Apoptosis in Fungal Development and Ageing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

DIANA BRUST, ANDREA HAMANN, HEINZ D. OSIEWACZ

5 Communication of Fungi on Individual, Species, Kingdom, and Above

Kingdom Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
URSULA KÜES, MÓNICA NAVARRO-GONZÁLEZ

6 Yeast Killer Toxins: Fundamentals and Applications . . . . . . . . . . . . . . . . . . . . . . . 107

FRIEDHELM MEINHARDT, ROLAND KLASSEN

7 Evolutionary and Ecological Interactions of Mould and Insects . . . . . . . . . . . . 133

MARKO ROHLFS, MONIKA TRIENENS, ULRIKE FOHGRUB, FRANK KEMPKEN

8 Endophytic Fungi, Occurrence and Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

DANIELA WEBER

9 Fungal Origin of Ergoline Alkaloids Present in Dicotyledonous Plants

(Convolvulaceae) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
ECKHARD LEISTNER, ULRIKE STEINER

10 Secondary Metabolites of Basidiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

ANJA SCHÜFFLER, TIMM ANKE

11 Identification of Fungicide Targets in Pathogenic Fungi . . . . . . . . . . . . . . . . . . . 233

ANDREW J. FOSTER, ECKHARD THINES

12 Helminth Electron Transport Inhibitors Produced by Fungi . . . . . . . . . . . . . . . 247

ROKURO MASUMA, KAZURO SHIOMI, SATOSHI ŌMURA

13 Cyclic Peptides and Depsipeptides from Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

HEIDRUN ANKE, LUIS ANTELO
xvi Contents

14 Fungal Genome Mining and Activation of Silent Gene Clusters . . . . . . . . . . 297

AXEL A. BRAKHAGE, SEBASTIAN BERGMANN, JULIA SCHUEMANN, KIRSTIN
SCHERLACH, VOLKER SCHROECKH, CHRISTIAN HERTWECK

15 Non-Ribosomal Peptide Synthetases of Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

KATRIN EISFELD

16 Biosynthesis of Fungal Polyketides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

JULIA SCHUEMANN, CHRISTIAN HERTWECK

17 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis . . . . . . 353

ROLF GEISEN, MARKUS SCHMIDT-HEYDT

18 Genetic and Metabolic Engineering in Filamentous Fungi . . . . . . . . . . . . . . . . . 377

JOCHEN SCHMID, ULF STAHL, VERA MEYER

Biosystematic Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

List of Contributors

HEIDRUN ANKE
(e-mail: anke@ibwf.de, Tel.: +49-631-31672-10, Fax: +49-631-31672-15)
Institute for Biotechnology and Drug Research, IBWF e.V., Erwin-Schroedinger-
Strasse 56, 67663 Kaiserslautern, Germany

TIMM ANKE
(e-mail: anke@rhrk.uni-kl.de, Tel.: +49-631-205-3046, Fax: +49-631-205-2999)
Institute for Biotechnology and Drug Research, IBWF e.V., Erwin-Schroedinger-
Strasse 56, 67663 Kaiserslautern, Germany

LUIS ANTELO
(e-mail: antelo@ibwf.de, Fax: +49-631-31672-15)
Institute for Biotechnology and Drug Research, IBWF e.V., Erwin-Schroedinger-
Strasse 56, 67663 Kaiserslautern, Germany

SEBASTIAN BERGMANN
Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research
and Infection Biology (HKI), and Friedrich Schiller University, Beutenbergstrasse 11a,
07745 Jena, Germany

AXEL A. BRAKHAGE
(e-mail: axel.brakhage@hki-jena.de, Tel.: +49-3641-65-6601, Fax: +49-3641-65-6825)
Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research
and Infection Biology (HKI), and Friedrich Schiller University, Beutenbergstrasse 11a,
07745 Jena, Germany

DIANA BRUST
(e-mail: brust@bio.uni-frankfurt.de)
Institute for Molecular Biosciences, Department of Biosciences and Cluster of
Excellence Macromolecular Complexes, Goethe-University, Max-von-Laue-Strasse 9,
60438 Frankfurt, Germany

KATRIN EISFELD
(e-mail: eisfeld@ibwf.de, Tel.: +49-631-31672-0, Fax: +49-631-31672-15)
Institute of Biotechnology and Drug Research, Erwin-Schrödinger-Strasse 56, 67663
Kaiserslautern, Germany

SKANDER ELLEUCHE
Abteilung Genetik eukaryotischer Mikroorganismen, Institut für Mikrobiologie und
Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen,
Germany
xviii List of Contributors

INES ENGH
(e-mail: ines.engh@rub.de, Tel.: +49-234-3224974, Fax: +49-234-3214184)
Lehrstuhl für Allgemeine und Molekulare Botanik, Fakultät für Biologie, Ruhr-
Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany

ULRIKE FOHGRUB
Botanical Institute, Department of Plant Genetics and Molecular Biology, Christian-
Albrechts-University of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany

ANDREW J. FOSTER
(e-mail: foster@ibwf.de, Tel.: +49-631-2054063, Fax: +49-631-3167215)
Institut für Biotechnologie und Wirkstoff-Forschung e.V./Institute for Biotechnology
and Drug Research, Erwin-Schrödinger-Strasse 56, 67663 Kaiserslautern, Germany

ROLF GEISEN
(e-mail: Rolf.Geisen@mri.bund.de, Tel.: +49-721-6625-450, Fax: +49-721-6625-453)
Max Rubner Institute, Federal Research Institute of Nutrition and Food, Haid-und-
Neu-Strasse 9, 76131 Karlsruhe, Germany

ANDREA HAMANN
(e-mail: a.hamann@bio.uni-frankfurt.de, Tel.: +49-69-798-29548, Fax: +49-69-798-
29435)
Institute for Molecular Biosciences, Department of Biosciences and Cluster of
Excellence Macromolecular Complexes, Goethe-University, Max-von-Laue-Strasse 9,
60438 Frankfurt, Germany

CHRISTIAN HERTWECK
(e-mail: christian.hertweck@hki-jena.de, Tel.: +49-3641-5321100/01, Fax: +49-3641-
5320408)
Leibniz Institute for Natural Product Research and Infection Biology, HKI, and
Friedrich Schiller University, Beutenbergstrasse 11a, 07745 Jena, Germany

FRANK KEMPKEN
(e-mail: fkempken@bot.uni-kiel.de, Tel.: +49-431-880-4274, Fax: +49-431-880-4248)
Botanical Institute, Department of Plant Genetics and Molecular Biology, Christian-
Albrechts-University of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany

ROLAND KLASSEN
(e-mail: roland.klassen@uni-muenster.de, Fax: +49-251-83-38388)
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-
Universität Münster, Corrensstrasse 3, 48149 Münster, Germany

ULRICH KÜCK
(e-mail: ulrich.kueck@ruhr-uni-bochum.de, Tel.: +49-0234-322-6212, Fax: +49-234-
32-14184)
Lehrstuhl für Allgemeine und Molekulare Botanik, Fakultät für Biologie, Ruhr-
Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany
 List of Contributors xix

URSULA KÜES
(e-mail: ukuees@gwdg.de, Tel.: +49-551-397024, Fax: +49-551-392705)
Division of Molecular Wood Biotechnology and Technical Mycology, Büsgen-
Institute, Georg-August-University Göttingen, Büsgenweg 2, 37077 Göttingen,
Germany

ECKHARD LEISTNER
(e-mail: eleistner@uni-bonn.de, Tel.: +49-228-73-3199, Fax: +49-228-73-3250)
Institut für Pharmazeutische Biologie, Rheinische Friedrich Wilhelm-Universität
Bonn, Nussallee 6, 53115 Bonn, Germany

ROKURO MASUMA
(e-mail: masuma@lisci.kitasato-u.ac.jp)
The Kitasato Institute for Life Sciences and Graduate School of Infection Control
Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

FRIEDHELM MEINHARDT
(e-mail: meinhar@uni-muenster.de, Tel.: +49-251-83-39825, Fax: +49-251-83-38388)
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-
Universität Münster, Corrensstrasse 3, 48149 Münster, Germany

VERA MEYER
University of Technology Berlin, Department of Microbiology and Genetics, Gustav-
Meyer-Allee 25, 13355 Berlin, Germany; and Leiden University, Institute of Biology,
Research Group for Molecular Microbiology, Wassenaarseweg 64, 2333 AL Leiden,
The Netherlands

MÓNICA NAVARRO-GONZÁLEZ
(e-mail: mnavarr@gwdg.de, Fax: +49-551-392705)
Division of Molecular Wood Biotechnology and Technical Mycology, Büsgen-
Institute, Georg-August-University Göttingen, Büsgenweg 2, 37077 Göttingen,
Germany

NICOLE NOLTING
Abteilung Genetik eukaryotischer Mikroorganismen, Institut für Mikrobiologie und
Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen,
Germany

MINOU NOWROUSIAN
Lehrstuhl für Allgemeine und Molekulare Botanik, Fakultät für Biologie, Ruhr-
Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany

SATOSHI ŌMURA
(e-mail: omura-s@kitasato.or.jp, Tel.: +81-3-5791-6101, Fax: +81-3444-8360)
The Kitasato Institute for Life Sciences and Graduate School of Infection Control
Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
xx List of Contributors

HEINZ D. OSIEWACZ
(e-mail: osiewacz@em.uni-frankfurt.de, Tel.: +49-69-798-24827, Fax: +49-69-798-24822)
Institute for Molecular Biosciences, Department of Biosciences and Cluster of
Excellence Macromolecular Complexes, Goethe-University, Max-von-Laue-Strasse 9,
60438 Frankfurt, Germany

STEFANIE PÖGGELER
(e-mail: spoegge@gwdg.de, Tel.: +49-551-3913930, Fax: +49-551-3910123)
Abteilung Genetik eukaryotischer Mikroorganismen, Institut für Mikrobiologie und
Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen,
Germany

MARKO ROHLFS
Zoological Institute, Department of Animal Ecology, Christian-Albrechts-University
of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany

KIRSTIN SCHERLACH
Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection
Biology (HKI), and Friedrich Schiller University, Beutenbergstrasse 11a, 07745 Jena,
Germany

JOCHEN SCHMID
(e-mail: j.schmid@lb.tu-berlin.de, Tel.: +49-30-314-72750, Fax: +49-30-314-72922)
University of Technology Berlin, Department of Microbiology and Genetics, Gustav-
Meyer-Allee 25, 13355 Berlin, Germany

MARKUS SCHMIDT-HEYDT
(e-mail: Markus.Schmidt-Heydt@MRI.Bund.de, Tel.: +49-721-6625-450, Fax: +49-721-
6625-453)
Max Rubner Institute, Federal Research Institute of Nutrition and Food, Haid-und-
Neu-Strasse 9, 76131 Karlsruhe, Germany

JULIA SCHUEMANN
Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection
Biology (HKI), and Friedrich Schiller University, Beutenbergstrasse 11a, 07745 Jena,
Germany

ANJA SCHÜFFLER
Institut für Biotechnologie und Wirkstoff-Forschung e.V./Institute for Biotechnology
and Drug Research, Erwin-Schroedinger-Strasse 56, 67663 Kaiserslautern, Germany

KAZURO SHIOMI
(e-mail: shiomi@lisci.kitasato-u.ac.jp)
The Kitasato Institute for Life Sciences and Graduate School of Infection Control
Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

ULF STAHL
(e-mail: Ulf.Stahl@lb.tu-berlin.de, Tel.: +49-30-314-72750, Fax: +49-30-314-72922)
University of Technology Berlin, Department of Microbiology and Genetics, Gustav-
Meyer-Allee 25, 13355 Berlin, Germany
 List of Contributors xxi

ULRIKE STEINER
(e-mail: u-steiner@uni-bonn.de)
Institut für Nutzpflanzenwissenschaften und Ressourcenschutz (INRES), Rheinische
Friedrich Wilhelm-Universität Bonn, Nussallee 9, 53115 Bonn, Germany

ECKHARD THINES
(e-mail: thines@ibwf.de, Tel.: +49-631-316720, Fax: +49-631-3167215)
Institut für Biotechnologie und Wirkstoff-Forschung e.V./Institute for Biotechnology
and Drug Research, Erwin-Schrödinger-Strasse 56, 67663 Kaiserslautern, Germany

MONIKA TRIENENS
Zoological Institute, Department of Animal Ecology, Christian-Albrechts-University
of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany

DANIELA WEBER
(e-mail: weber-daniela@gmx.de, Tel.: +49-68-94-590-950, Fax: +49-68-94-590-95-275)
Institute of Biotechnology and Drug Research, Erwin-Schrödinger-Strasse 56, 67663
Kaiserslautern, Germany; present address: MIP International Pharma Research
GmbH, Mühlstrasse 50, 66386 St. Ingbert, Germany

ROLAND W.S. WEBER

(e-mail: Roland.Weber@lwk-niedersachsen.de, Tel.: +49-4162-6016-133, Fax: +49-
4162-6016-99133)
Obstbau Versuchs- und Beratungszentrum/Fruit Research and Advisory Centre
(OVB) Jork, Moorende 53, 21635 Jork, Germany
1 Recent Developments in the Molecular Taxonomy of Fungi

ROLAND W.S. WEBER1

CONTENTS schemes, depending on which features they

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 regarded as relevant and at which taxonomic level.
II. Non-Fungal Organisms Studied The past two decades have witnessed a revolu-
by Mycologists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 tion in the taxonomy of fungi because it became
A. Slime Moulds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 possible to generate and analyse homologous
B. Plasmodiophora and Related Species . . . . . . . . . 3
C. Straminipila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 DNA sequences of functionally conserved genes
D. Haptoglossa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 (initially mainly ribosomal DNA sequences) on a
III. The ‘Basal Fungi’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 routine basis, leading to a more ‘objective’ com-
A. Microsporidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 parison of taxa. Initial attempts at molecular phy-
B. Chytrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 logeny created considerable complications because
C. Zygomycete-Type Fungi . . . . . . . . . . . . . . . . . . . . . . . 6
D. Glomeromycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 each phylogenetic tree was usually based on the
IV. Ascomycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 analysis of a single gene, and widely different phy-
A. Taphrinomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 logenies could result if different genes were used.
B. Saccharomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 The situation around the turn of the millennium
C. Pezizomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
V. Basidiomycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
and in the following years was therefore compara-
A. Pucciniomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 ble to a football game with shifting goal-posts, as
B. Ustilaginomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . 11 especially those involved in teaching fungal taxon-
C. Agaricomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 omy will recall with horror. Several publications
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 also reflect the fluid state of the discipline at that
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
time, especially The Mycota VIIA and VIIB
(McLaughlin et al. 2001) and the ninth edition of
Ainsworth and Bisby’s Dictionary of the Fungi
(Kirk et al. 2001). These works have become out-
I. Introduction dated quite rapidly in terms of taxonomic con-
cepts, though not, of course, in their valuable
Taxonomy is the science of classification, i.e. the information on general biological features of the
grouping of organisms into defined categories fungi. The third edition of Introduction to Fungi
(taxa). Ideally, a classification scheme should be (Webster and Weber 2007), although principally
‘natural’ in the sense that all organisms in a given intended to be used as a textbook pursuing a
taxon should be related to each other by common holistic approach to the fungi rather than as a
ancestry. Traditionally, however, this was not neces- taxonomic work, also had to be based on the tax-
sarily the case with fungi because taxonomic onomy of the period. By the time of manuscript
approaches based on morphological and microscop- submission in March 2006, the outlines of certain
ic characters, later augmented by ultrastructural and ‘natural’ groups of fungi had become apparent,
biochemical features, did not always distinguish be- but these could neither be delimited clearly, nor
tween homologies and analogies. Traditional taxo- was it possible to assign formal names to many of
nomies were arbitrary also in the sense that different them for lack of existence, validation or general
taxonomists proposed widely differing classification acceptance.
This state of transition led a large consortium
1
Obstbau Versuchs- und Beratungszentrum/Fruit Research of fungal taxonomists to join forces and develop a
and Advisory Centre (OVB) Jork, Moorende 53, 21635 Jork, classification scheme based on multi-gene phylo-
Germany; e-mail: roland.weber@lwk-niedersachsen.de genies in the hope that this would provide a

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
2 Roland W.S. Weber
sound, permanent framework. Following the es-
tablishment of a communications platform (‘deep
hypha’), the ‘AFTOL’ (assembling the fungal tree
of life) project supported most of the immense
amount of DNA sequencing work and data analy-
sis (Blackwell et al. 2006). Both undertakings were
funded by the United States National Science
Foundation (NSF). The entire November/Decem-
ber 2006 issue of Mycologia was dedicated to
reports of the results of these efforts, and a classi-
fication scheme synthesising the main findings
was published by Hibbett et al. (2007). These
authors emphasised the broad support and input
which their scheme had received from numerous
taxonomists, and called on the worldwide myco-
logical community to adopt their unified system
with generally accepted taxon names in future.
To this end, many of the names of higher taxa
proposed by Hibbett et al. (2007) are based on
concepts readily recognised by most mycologists,
which greatly facilitates the usage of their scheme.
An arguable exception is the re-naming of higher
taxa after key species, which would replace e.g.
Urediniomycetes by Pucciniomycotina or Hyme-
nomycetes by Agaricomycotina. It is reassuring to
see that the introduction of these terms is an
ongoing process with occasional inconsistencies
even in publications by leading ‘AFTOL’ authors.
Therefore, the main purpose of the present
chapter is to give a brief overview of the current
taxonomic concept, highlighting changes from
equivalent taxonomic groupings delimited in ear-
lier schemes represented by Webster and Weber
(2007), McLaughlin et al. (2001) and Kirk et al.
(2001). At the same time, the likely stability of the
‘AFTOL’ system is evaluated. Since work by this
consortium did not cover organisms unrelated to
Fungi but nonetheless studied by mycologists,
their current phylogenetic and taxonomic place-
ment is also summarised. Finally, there is a brief
consideration of problems and potential of
current fungal taxonomy in mycology teaching.
II. Non-Fungal Organisms Studied
by Mycologists
Genera such as Phytophthora, Pythium, Peronos-
pora, Plasmopara or Plasmodiophora are so
intrinsically linked with the history of mycology
and fungal plant pathology that they continue to
be studied in mycological laboratories and to be
taught in mycology lectures and practical classes.
Slime moulds and other non-fungal protists will
be encountered by mycologists during forays.
Protists are subjected to two mutually incompati-
ble classification systems: a zoological one gov-
erned by the International Code of Zoological
Nomenclature and a mycological one according
to the International Code of Botanical Nomencla-
ture. The resulting confusion is multiplied by the
rapid discovery of new protist species and new
relationships between existing taxa by molecular
phylogenetic approaches. Adl et al. (2005, 2007),
recognising that neither of the two Codes provides
a stable classification system for these organisms,
broke free of both and established a scheme which
retains as many of the commonly known names
as possible. This move is supported by many
protozoologists.
Some five or six kingdom-sized super-groups
of eukaryotes can be resolved by current phyloge-
netic studies based on multi-gene analyses and
modern analytical methods (Simpson and Roger
2004; Keeling et al. 2005). All but one of
these harbour organisms studied by mycologists
(Fig. 1.1). An overview of formal and informal
names of relevant higher taxa is given in Fig. 1.2.
A. Slime Moulds
Slime moulds may exist vegetatively as amoebae
(cellular slime moulds) or as multinucleate
plasmodia (plasmodial slime moulds). Both ingest
particulate food by phagocytosis. Reproduction is
by means of single-spored or multi-spored struc-
tures termed sporocarps or sorocarps, respectively.
Flagellate stages (swarmers) bearing two whiplash-
type flagella may be present in the life cycle of
certain groups (myxogastrids, dictyostelids).
The separation of a small group, the acrasids,
from the bulk of slime moulds has long been
recognised on the basis of phylogenetic studies
(Baldauf et al. 2000) and finds its morphological
manifestation in the shape of the amoeba which
produces a single lobed (lobose) anterior pseudo-
podium in acrasids, as opposed to the pointed
(filose) pseudopodia emitted by amoebae of all
other slime moulds. Acrasids are now grouped
among the Excavata. All other slime moulds, in-
cluding both cellular and plasmodial forms, be-
long to three phylogenetically related groups
 Recent Developments in the Molecular Taxonomy of Fungi
Fig. 1.1. A hypothetical phylogenetic tree of eukaryotes
showing five ‘supergroups’, based on molecular phylo-
genies, other molecular characters and morphological
and biochemical evidence. Dotted lines indicate relation-
ships resolved on preliminary evidence only, whereas
within the Amoebozoa (Fig. 1.1). Dictyostelid
slime moulds are cellular forms, represented by
the well-known Dictyostelium discoideum with
its cAMP-mediated aggregation of thousands of
amoebae into a pseudoplasmodium, which goes
on to form a slug and ultimately a sorocarp
(see Kessin 2001). Protostelid slime moulds may
form filose amoebae or small plasmodia, whereas
in myxogastrid forms such as Fuligo septica or
Physarum polycephalum, the plasmodium is dom-
inant, amoebae being reduced in the life cycle
to a brief period following spore germination
(see Webster and Weber 2007).
3
relationships were left unresolved where there was no
evidence of branching order. Groups traditionally stud-
ied by mycologists are printed in capital letters.
Re-drawn from Keeling et al. (2005), with permission
from Elsevier
B. Plasmodiophora and Related Species
Plasmodiophora brassicae is the cause of club root
of brassicas. Infection is initiated when a zoospore
with two whiplash-type flagella encysts on a root
hair. The penetration mechanism is highly char-
acteristic in that a bullet-shaped stylet is forced
by turgor pressure from the spore cyst through
the root cell wall, injecting a small wall-less
amoeba into the host cytoplasm. Plasmodia capa-
ble of phagocytosis develop from this amoeba and
migrate deeper into the root tissue, breaking
through plant cell walls along their way.
4 Roland W.S. Weber
Fig. 1.2. Organisms not belonging to the Fungi yet tradi-
tionally studied by mycologists, in a mycological system
(Webster and Weber 2007) and in the protistological clas-
sification scheme of Adl et al. (2005). Organisms grouped
within a phylum but already suspected at the time of being
phylogenetically unrelated to each other are separated
by dotted boxes. Orders united by a bracket are currently
grouped together in Peronosporales by many authors
Archibald and Keeling (2004) and others have
provided evidence that Plasmodiophora and allied
genera (Spongospora, Polymyxa) are related to
Cercozoan protists. Adl et al. (2005) have also
placed plasmodiophorids among the Cercozoa
within the ‘kingdom’ Rhizaria (see Fig. 1.1).
C. Straminipila
Organisms possessing cellulose-containing cell
walls, an inner mitochondrial membrane folded
into tubular rather than plate-like cristae, morpho-
logically recognisable Golgi stacks, biflagellate het-
erokont zoospores (i.e. with one flagellum of the
tinsel and the other of the whiplash type), and a
lysine biosynthetic pathway via a,e-diaminopimelic
acid (DAP) rather than a-aminoadipic acid (AAA),
are accommodated in a kingdom named Chromista
by workers emphasising the lack of chlorophyll b in
its photosynthetic members, or Straminipila by
others highlighting the tinsel-type flagellum with
its uniquely complex architecture. Straminipila/
Chromista are now grouped together with Alveolata
(dinoflagellates, ciliates, and other taxa; Fast et al.
2002) in the ‘kingdom’ Chromalveolata (Simpson
and Roger 2004; Keeling et al. 2005).
Several phyla within the Straminipila are of
relevance to mycology. The phylum Labyrinthu-
lomycota contains marine organisms in which
individual vegetative cells produce a network of
slime tracks. In addition, each cell is surrounded
by a wall comprising a Golgi-derived L-galactose
polymer. In Labyrinthulales, the cells move within
their slime tracks, whereas in Thraustochytriales
the slime track forms a rhizoid-like network at the
base of a thallus-like structure (see Webster and
Weber 2007). Both orders produce heterokont
zoospores.
Hyphochytriomycota resemble chytrid fungi
in forming thalli comprised of one or more spo-
rangia linked by rhizoids. The zoospore contains
only a forward-directed tinsel flagellum, the back-
ward-pointing whiplash flagellum having been
lost during the course of evolution.
By far the most important group of stramini-
pilous organisms from a mycological perspective
is the phylum Oomycota. Included here are organ-
isms with heterokont zoospores and oogamous
sexual reproduction, e.g. filamentous water
moulds such as the Saprolegniales (Saprolegnia,
Achlya), thallic aquatic saprotrophs or parasites
of algae and other organisms, and filamentous
species adapted to the terrestrial environment.
Terrestrial straminipilous fungi have traditionally
been separated into Pythiales (saprotrophic forms
and necrotrophic pathogens especially of plants),
Peronosporales and Sclerosporaceae (obligately
biotrophic downy mildews of dicotyledonous
plants and grasses, respectively). However, several
recent phylogenetic analyses point towards inter-
calations between these taxa (Riethmüller et al.
2002; Villa et al. 2006; Thines et al. 2008) so that
they may all eventually be united in one group
(e.g. Peronosporales), at the possible exclusion of
Albugo (Hudspeth et al. 2003). The collateral
abandonment or revision of well-known genera
such as Pythium, Phytophthora or Peronospora
 Recent Developments in the Molecular Taxonomy of Fungi 5

has not yet been completed. In view of the ongo-

ing trend to name higher taxa on the basis of
exemplar genera, it may be deemed necessary
to replace the term Oomycota/Oomycetes by
Peronosporomycetes (Dick 2001).

D. Haptoglossa

Haptoglossa spp. infect rotifers or nematodes by a

forceful injection mechanism very similar to that
of Plasmodiophora (see above), although a walled
sporidium instead of a wall-less amoeba enters the
living host. Further, zoospores of Haptoglossa,
where formed, appear to be anisokont, i.e. with
two whiplash-type flagella of unequal length, like
those of Plasmodiophora. Nonetheless, molecular
phylogenetic studies have shown Haptoglossa spp.
to be members of Straminipila, and indeed to
belong to the Peronosporomycetes (Hakariya
et al. 2007). Haptoglossa had also been grouped
there by Adl et al. (2005) and, more intuitively,
by earlier workers.

III. The ‘Basal Fungi’

James et al. (2006a) published a 70-author paper

about the trunk of the fungal phylogenetic tree
based on analyses of six functionally conserved
genes, i.e. the nuclear ribosomal DNA operon
(18S-, 28S-, 5.8S-rDNA), the ribosomal elongation
factor gene EF1a, and the RNA polymerase II
subunit genes RPB1 and RPB2. As a result of this
fundamental work and numerous other contribu-
tions, the taxonomy of the basal fungi was mod-
ified extensively by Hibbett et al. (2007), as
summarised in Fig. 1.3. The two established
phyla, Chytridiomycota and Zygomycota, were
broken into a total of eight groups, to which a
further phylum, the Microsporidia, was added.
A. Microsporidia
The inclusion of the Microsporidia within the
Fungi has been highly controversial until very
recently (e.g. Keeling et al. 2000; Tanabe et al.
2002), and the continued use of the zoological
term ‘Microsporidia’ is a reminder of that debate.
Included in this phylum are the most basal fungi
according to current phylogenetic knowledge.
Fig. 1.3. Classification of the lower fungi according to the
current ‘AFTOL’ scheme (Hibbett et al. 2007) in comparison
with Webster and Weber (2007) summarising earlier
approaches. Groups highlighted by an asterisk were men-
tioned by these authors but not discussed in detail. Supple-
mentary data from Kirk et al. (2001) are indicated in square
brackets
These organisms are obligate parasites of animals,
infecting their host cells by injection of a small
protoplast from a spore through a tube initially
coiled up inside the spore, then extruded by os-
motic pressure (Keeling and Fast 2002). Such an
infection mechanism is reminiscent of that found
in Plasmodiophora (Cercozoa) or Haptoglossa
(Chromalveolata, Straminipila) with which Micro-
sporidia are not related. Instead, their closest rel-
ative in the analysis by James et al. (2006a) was the
chytrid fungus Rozella allomycis, an endoparasite
of Allomyces. Whereas R. allomycis possesses a
posteriorly uniflagellated zoospore typical of the
chytrids, Microsporidia do not, indicating that
6 Roland W.S. Weber
the flagellum must have been lost during the
course of evolution. Microsporidia show an accel-
erated rate of DNA sequence evolution as com-
pared to other fungi, and this may be explained by
the loss of function of many genes due to an
increased reliance upon the host’s metabolic ma-
chinery (Inagaki et al. 2004). Such trends may
cause ‘long branch attractions’, i.e. the false
grouping-together of unrelated organisms at the
base of phylogenetic schemes. Therefore, the
trunk of the fungal tree and the circumscription
of the phylum Microsporidia are still somewhat
diffuse at present.
B. Chytrids
The original phylum Chytridiomycota contained
all true Fungi with zoospores bearing a single
posterior whiplash-type flagellum, or several such
flagella (as in the anaerobic rumen fungi). The
thallus is either holocarpic, consisting of a sac-
like structure which later converts to a zoosporan-
gium, or eucarpic, i.e. sporangia are formed from
rhizoids or a rhizomycelium. Monophyly of the
Chytridiomycota could not be upheld on the
basis of the studies by James et al. (2006a, b) and
Hibbett et al. (2007). Instead, this taxon was
retained as a more narrowly defined group, and
two additional phyla were created: Neocallimasti-
gomycota and Blastocladiomycota (Fig. 1.3). The
Blastocladiomycota were identified as a sister
group to the basal non-flagellated fungi formerly
called Zygomycota, and to higher fungi (Ascomy-
cota, Basidiomycota).
Nonetheless, there are still some escapees from
a unified chytrid taxonomy, e.g. R. allomycis with
an affinity for Microsporidia as mentioned above,
and Olpidium brassicae which groups with Basidio-
bolus ranarum close to the Entomophthorales
(Zygomycota). Placement of Basidiobolus in the
vicinity of Entomophthorales is a relief to teaching
mycologists because this fungus shares so many
key biological features with the insect-pathogenic
Entomophthorales that its inclusion in the Chytri-
diomycota as suggested by preliminary phylogenet-
ic studies (e.g. Berbee and Taylor 2001) did not
make any biological sense (Webster and Weber
2007). All the more curious is the current associa-
tion of O. brassicae, a seemingly typical chytrid,
with the Entomophthorales; it will be interesting
to see whether this remains its final resting place.
Rhizophlyctis and Caulochytrium also remain to be
accommodated at ordinal level (Hibbett et al. 2007),
and the establishment of further chytrid orders
therefore cannot be ruled out.
James et al. (2006a) discussed the number of
occasions on which the flagellum may have been
lost in the course of fungal evolution, and counted
at least four; once in Rozella, once in Hyaloraphi-
dium curvatum, formerly thought to be derived
from a marine alga (Ustinova et al. 2000), and at
least twice en route to the zygomycetous fungi.
C. Zygomycete-Type Fungi
Zygomycetous fungi reproduce sexually by zygo-
spores arising from suspensors which are usually
isogamous. Asexual reproduction is typically by
sporangiospores arising from internal cleavage of
protoplasm, or by single-spored sporangia or even
true conidia (Webster and Weber 2007). Fungi for-
merly included in the phylum Zygomycota present
one of the greatest challenges to current phylogeny-
based classification because several recent studies
have given conflicting results of monophyly or
polyphyly (Tanabe et al. 2004; Liu et al. 2006;
White et al. 2006). Mindful of these uncertainties,
Hibbett et al. (2007) broke up the Zygomycota into
four subphyla, but held open the possibility that a
revised Zygomycota of more restricted circum-
scription might be re-established and validated in
future. This would be highly desirable, especially
from the perspective of mycology teaching.
The subphylum Mucoromycotina includes the
largest order, Mucorales, together with Mortierel-
lales and Endogonales. The former two contain
mainly saprotrophic members, the latter faculta-
tively ectomycorrhizal species forming ‘pea truf-
fles’ in which zygospores are clustered together in
subterranean fruit-bodies. Webster and Weber
(2007) did not describe Endogonales in detail
and included Mortierella and its allies in Mucor-
ales on the grounds of unifying biological themes
while being aware of their possible status as a
separate order. Two other subphyla, the mostly
entomopathogenic Entomophthoromycotina and
the Zoopagomycotina (pathogens of animals
and fungi), were treated as orders by Webster
and Weber (2007); that change to sub-phylum is
easily accommodated.
A difficult case is presented by the Kickxellomy-
cotina, a group formerly regarded as ‘trichomycetes’.
 Recent Developments in the Molecular Taxonomy of Fungi
Included here are biologically heterogeneous sapro-
trophs and mycoparasites (Kickxellales, Dimargari-
tales) as well as commensals or weak parasites of
insect guts (Harpellales and Asellariales, i.e. tricho-
mycetes sensu stricto). Other orders formerly
regarded as trichomycetes, especially Eccrinales and
Amoebidiales, were relegated to protozoa (Cafaro
2005), where they were classified among Ichthyo-
sporea by Adl et al. (2005; see Fig. 1.1). Nonetheless,
the Kickxellomycotina represent a likely candidate
for substantial future taxonomic rearrangements
among zygomycete-type fungi.
D. Glomeromycota
Formerly included in the order Glomales among
the Zygomycota (Morton and Benny 1990), this
group of obligately symbiotic fungi associated
with vesicular-arbuscular mycorrhiza was given
the status of a phylum, Glomeromycota (Schüssler
et al. 2001). This placement has won widespread
support as being monophyletic. The only known
non-mycorrhizal member is Geosiphon pyriformis,
which contains intracellular cyanobacteria as
symbionts. Because glomeralean fungi cannot be
grown in pure culture, species are difficult to
define, and considerable re-arrangements below
the phylum level may well have to be performed
in future (Redecker and Raab 2006). Among all
basal fungi, Glomeromycota are the closest known
relatives of Basidiomycota and Ascomycota (James
et al. 2006a).
IV. Ascomycota
The subkingdom Dikarya, referring to the presence
of at least a short dikaryotic phase in the life cycle,
was erected by Hibbett et al. (2007) to accommodate
Ascomycota and Basidiomycota, thereby reflecting
their close phylogenetic relationship. It is debatable
whether this subkingdom is necessary from a taxo-
nomic point of view if there is no counterpart to
group together the ‘lower fungi’. The retention of
Ascomycota and Basidiomycota as phyla is wel-
come as these terms are in universal use (e.g. Kirk
et al. 2001). Within the phylum Ascomycota, three
subphyla are currently recognised and are dis-
cussed in their turn. A summary of current classes
and orders of Ascomycota is given in Fig. 1.4.
7
A. Taphrinomycotina
Nishida and Sugiyama (1994) gave the provisional
name ‘Archiascomycetes’ to this most basal group
of Ascomycota, but Hibbett et al. (2007) accepted
the term Taphrinomycotina, originally coined by
Eriksson and Winka (1997), to describe the same
taxonomic grouping. Included are the yeasts Schi-
zosaccharomyces and Saitoella, the plant patho-
gens Taphrina and Protomyces, the mammal
pathogen Pneumocystis, and the unusual apothe-
cial fungus Neolecta (Sugiyama et al. 2006). There
is still some controversy over the monophyly of
this group even after numerous phylogenetic
studies, but also because there are so few obvious
morphological characters uniting its members.
One of these is that ascogenous hyphae are
lacking, even in the filamentous Neolecta, and
that ascospores are capable of germinating by
budding. Taphrina is believed to be close to the
evolutionary origin of both Ascomycota and Basi-
diomycota, and in this context it is tantalising that
both yeasts and filamentous fungi are found in the
Taphrinomycotina, thereby leaving the question
open to future discussion as to which state is
ancestral in the Dikarya.
B. Saccharomycotina
This subphylum, equivalent to the ‘Hemiascomy-
cetes’ (Kurtzman and Sugiyama 2001; Webster
and Weber 2007), contains most of the ascomy-
cete yeasts. It is phylogenetically well-defined with
only one order, Saccharomycetales (James et al.
2006a; Suh et al. 2006). Considerable rearrange-
ments at the genus level are certain as more DNA
sequences become available and the unreliable
nature of physiological tests becomes more obvi-
ous. Pichia and Candida are extreme examples of
polyphyletic genera within the Saccharomycotina.
C. Pezizomycotina
With over 30 000 species, this is by far the largest
group of Ascomycota, and it is regarded as mono-
phyletic (Spatafora et al. 2006). It is equivalent in its
circumscription to the ‘filamentous ascomycetes’
(Alexopoulos et al. 1996) or ‘Euascomycetes’
(Kurtzman and Sugiyama 2001). Inevitably, there
have been major rearrangements within the sub-
phylum, and these will continue for years to come.
8 Roland W.S. Weber
Fig. 1.4. Classification of the Ascomycota according to
the current ‘AFTOL’ scheme (Hibbett et al. 2007) in
comparison with the state of knowledge in 2005 as used
by Webster and Weber (2007). Supplementary data from
Most groups within the Pezizomycotina have now
been accommodated in classes, although there are
still some nomadic units, among them families
including important or widely known fungi
such as Magnaporthaceae (Magnaporthe grisea),
Kirk et al. (2001) are indicated in square brackets.
Question marks indicate taxonomic positions incertae
sedes, the letter L the inclusion of lichenised forms in the
order
Glomerellaceae (Glomerella, Colletotrichum), and
Geoglossaceae (Geoglossum, Trichoglossum). Fur-
ther, several established orders cannot yet be an-
chored securely in higher taxa and must remain
incertae sedes at present. A recent phylogenetic
 Recent Developments in the Molecular Taxonomy of Fungi
analysis of classes within the Pezizomycotina may
be found in Spatafora et al. (2006). Clearly, there is
ample scope for further rearrangements which
questions the stability of the system proposed by
Hibbett et al. (2007).
The most basal classes of Pezizomycotina are
the small group Orbiliomycetes and the larger Pezi-
zomycetes, each with a single order. These groups
are well-separated from a ‘crown’ of other pezizo-
mycotinoid ascomycetes (James et al. 2006a). Com-
plex fruit-bodies (stromata) are absent, and asci are
arranged in apothecia in most cases, only rarely in
hypogeous ascocarps (truffles) or in cleistothe-
cium- or perithecium-like forms. Some progress is
being made with the classification of Pezizales at the
sub-ordinal level (Hansen and Pfister 2006).
A notoriously difficult group is the class
Dothideomycetes, formerly known as Loculoasco-
mycetes. The multi-gene analysis by Schoch et al.
(2006) confirmed the existence of two previously
suspected groups, now called Dothideomycetidae
and Pleosporomycetidae, but left several orders
incertae sedes. Lichenised fungi were not included
in that analysis, and these are likely to complicate
the matter further.
A group with a similarly high degree of mor-
phological and biological variation is the class
Eurotiomycetes (formerly Plectomycetes; Geiser
and LoBuglio 2001). ‘Core groups’ are Onygenales
(which form gymnothecia or cleistothecia and
comprise important human and animal pathogens)
and Eurotiales (containing Aspergillus and Penicil-
lium); a second clade identified by Geiser et al.
(2006) contains lichen-forming as well as non-
lichenised orders (Verrucariales, Chaetothyriales,
Pyrenulales).
Another phylogenetically defined but biologi-
cally heterogeneous group is the class Leotiomy-
cetes containing ‘inoperculate discomycetes’
(Helotiales) as their core group (Wang et al. 2006).
Although indicated by previous studies (Tsuneda
and Currah 2004), the confirmation of typical
gymnothecial fungi such as Myxotrichum and Pseu-
dogymnoascus as members of this class will come
as a surprise to many, as will the inclusion of
Erysiphales (powdery mildews) and the exclusion
of Geoglossaceae (earth-tongues). It will be a
challenge to teach Leotiomycetes, or give a written
account of them, in a taxonomic context!.
Sordariomycetes is the name now applied to
a large group formerly called Pyrenomycetes
(Samuels and Blackwell 2001). However, while
9
the name may have changed, most of the impor-
tant orders have been confirmed in an excellent
phylogenetic account by Zhang et al. (2006),
indicating areas of further work such as the need
to accommodate Magnaporthaceae and Glomerel-
laceae as well as several orders of uncertain
position.
Probably the most difficult phylogenetic prob-
lem within the Ascomycota is presented by liche-
nised fungi which make up almost all the species
within Lecanoromycetes, the largest class of Asco-
mycota, but are also found in Dothideomycetes,
Eurotiomycetes and in the smaller classes Lichi-
nomycetes and Arthoniomycetes. The compre-
hensive study by Miadlikowska et al. (2006) on
Lecanoromycetes is remarkable in being the first
major phylogenetic work to take account of
photobiont identity as a much-needed additional
taxonomic marker. Nonetheless, extensive future
rearrangements may be expected.
V. Basidiomycota
Almost all Basidiomycota can be assigned to one
of three broad subphyla (i.e. Pucciniomycotina,
Ustilaginomycotina, Agaricomycotina) erected by
Bauer et al. (2006) to replace the former classes
Urediniomycetes, Ustilaginomycetes and Hymeno-
mycetes, respectively. Currently homeless taxa are
Entorrhizales, a small group of smut-like patho-
gens of monocot roots formerly included among
the Ustilaginomycotina or a synonym, and Walle-
mia, a mitosporic mould of relevance as a food
spoilage fungus and in indoor situations (Hibbett
2006). A summary of the current taxonomic con-
cept by Hibbett et al. (2007) is presented in Fig. 1.5.
A. Pucciniomycotina
This subphylum, equivalent in its circumscription
to the Urediniomycetes (Kirk et al. 2001), has been
subjected to numerous taxonomic and phylogenet-
ic studies. Earlier schemes were summarised by
Swann et al. (2001) who recognised that the simple,
ascomycete-like architecture of hyphal septa
distinguished the Pucciniomycotina from that of
the other two major subphyla of Basidiomycota,
i.e. Ustilaginomycotina (membrane-bounded
septal caps) and Agaricomycotina (dolipore
septa). These and many other ultrastructural
10 Roland W.S. Weber
Fig. 1.5. Classification of the Basidiomycota according to
the current ‘AFTOL’ scheme (Hibbett et al. 2007) in com-
parison with data available in 2005 (Webster and Weber
and biochemical features are consistent with the
basal position within the Basidiomycota which
Pucciniomycotina occupy in many phylogenetic
analyses (Bauer et al. 2006). However, whilst the
range of genera to be included in this subphylum
had become evident, their grouping into higher
taxa was still tentative. Bauer et al. (2006) evalu-
ated over 30 years of research into ultrastructural
features, backed up by phylogenetic studies of
rDNA loci. Their classification scheme was sup-
ported by the comprehensive phylogenetic study
of Aime et al. (2006) and accepted by Hibbett
et al. (2007).
2007). Supplementary data from Kirk et al. (2001) and
McLaughlin et al. (2001) are indicated in square brackets.
Positions incertae sedes are indicated by question marks
The class Pucciniomycetes is by far the most
important one in this subphylum. The order
Pucciniales (formerly Uredinales) includes all
known rust fungi, obligately biotrophic plant
pathogens of which about 7000 species are
known. These have distinct monokaryotic and
dikaryotic phases which may alternate between
two taxonomically unrelated host plants, pro-
ducing up to five different spore stages in the
process (Webster and Weber 2007). An even
more extreme case of host alternation is pre-
sented by the Helicobasidiales (ca. 6 spp.)
which parasitise rust fungi in their haploid
 Recent Developments in the Molecular Taxonomy of Fungi
conidial phase (Tuberculina), whereas their
dikaryotic phase (Rhizoctonia-like as a sterile
mycelium, Helicobasidium when producing basi-
dia) causes violet root rot on a wide range of
terrestrial plants (Lutz et al. 2004). The Septoba-
sidiales (170 spp.) are dimorphic; yeast cells
produced from basidiospores can infect scale
insects where a mycelium is formed which
ultimately produces resupinate fruit-bodies out-
side the dead insect host.
The remaining seven classes contain fungi
with an astonishing diversity of life styles (plant-
pathogenic, mycoparasitic, aquatic, saprotrophic)
and diverse growth forms, as summarised in the
insightful discussion of biological and ultrastructural
features by Bauer et al. (2006). Yeasts of the
Rhodotorula and Sporobolomyces types occur in
the classes Cystobasidiomycetes (Cystobasidiales)
and in Microbotryomycetes (Sporidiales). Other
yeast genera are also found in these two classes.
Macroscopic fruit-bodies, where present, are stil-
boid, i.e. small, jelly-like and stalked with an
enlarged head. The class Microbotryomycetes
includes the Microbotryales, an order of fungi
formerly called ‘dicot Ustilago spp.’ and included
in the Ustilaginales because of their striking
biological and morphological similarities to the
true smut fungi (discussed by Webster and
Weber 2007).
Because most of the remaining classes and
orders are mono- or oligotypic, we may expect
the discovery of many more species especially
from tropical habitats, and this could destabilise
the current taxonomic system (Aime et al. 2006).
B. Ustilaginomycotina
This group of approximately 1500 species contains
the true smut fungi and their allies with unifying
biological features of saprotrophic haploid yeast
cells which copulate and establish a dikaryotic my-
celium as prerequisite for infection of host plants
(Webster and Weber 2007). Septal pores are com-
monly surrounded by simple membrane caps. De-
spite a relatively limited number of groups, the
taxonomy of the Ustilaginomycotina is still far
from settled. Recent advances in our understand-
ing both of ultrastructural features and rDNA-
based phylogenetics have led to a revised classifi-
cation of Ustilaginomycotina (Bauer et al. 1997;
Begerow et al. 1997) which has repeatedly been
11
modified (see Begerow et al. 2006). There are two
firmly established classes, Ustilaginomycetes and
Exobasidiomycetes (Hibbett et al. 2007). The
Entorrhizales were included by Begerow et al.
(2006) but excluded by Hibbett et al. (2007), where-
as the Malasseziales, a group of anamorphic yeasts
associated with warm-blooded animals, are incer-
tae sedes within the Ustilaginomycotina.
Members of the Ustilaginomycetes (Urocys-
tales, Ustilaginales) almost exclusively infect
monocots (grasses, sedges, rushes). No true haus-
toria are formed, but intracellular hyphae may be
present, surrounded by a thick sheath (Bauer et al.
1997). Infection culminates in the production of
thick-walled teliospores from which basidia arise.
The scorched appearance of infected, teliospore-
releasing plant tissue has given the smut fungi
their name. The class Exobasidiomycetes contains
a more diverse assemblage of fungi. Members of
the order Exobasidiales parasitise dicot trees and
shrubs, forming basidia without the teliospore
stage on the infected and often swollen host
surface. The electron-dense sheath around intra-
cellular hyphae of Ustilaginomycetes is reduced to
a few plaques in Exobasidium (Begerow et al.
2002). Confusingly, typical smut fungi are also
included in the Exobasidiomycetes, e.g. Tilletiales,
Entylomatales, and Georgefischeriales, in addition
to aquatic smuts (Doassansiales) and Tilletiopsis-
type yeasts (Entylomatales).
The conclusion we must draw is that typical
‘smut fungi’ are found in several phylogenetically
distant groups, including Microbotryales (Pucci-
niomycotina), Entorrhizomycetes (Basidiomycota
incertae sedes), and Ustilaginomycotina (Ustilagi-
nomycetes and Exobasidiomycetes). Further, the
taxonomy of Ustilaginomycetes is still in a state of
flux which even the comprehensive recent multi-
gene study by Begerow et al. (2006) has not alto-
gether consolidated.
C. Agaricomycotina
This subphylum, approximately synonymous with
the Hymenomycetes (Swann and Taylor 1993) or
Basidiomycetes (Kirk et al. 2001), provides the
most striking fruit-bodies of all fungi, and these
have been studied for centuries in a taxonomic con-
text. Unfortunately, fruit-body morphology has been
found in many cases to be a poor indicator of true
phylogenetic relationships, explaining the unusually
12 Roland W.S. Weber

high rate of rearrangements within the Agaricomy-
cotina in recent years. Hibbett (2006) and Hibbett
et al. (2007), summarising the current viewpoint of
AFTOL project members, subdivided the Agarico-
mycotina into three classes, i.e. Tremellomycetes,
Dacrymycetes, and Agaricomycetes, but this is un-
likely to remain the last word on the matter as
numerous orders remain incertae sedes.
In the past, fungi have been called heterobasi-
diomycetes or phragmobasidiomycetes if: (1)
their basidia possess highly differentiated sterig-
mata (epibasidia) or are even divided by trans-
verse or longitudinal septa, (2) basidiospores
germinate by budding in a yeast-like manner
or by producing ballistosporic conidia, and (3)
fruit-bodies, if present, are jelly-like and capable
of discharging basidiospores even after several
drying/rehydration cycles (Kirk et al. 2001; Wells
and Bandoni 2001; Webster and Weber 2007).
Unfortunately, these features, like many others,
are now known to be phylogenetically irrelevant,
and ‘heterobasidiomycetes’ may be found in Tre-
mellomycetes (Tremellales), Dacrymycetes
(Dacrymyces, Calocera) and certain orders of the
Agaricomycetes, such as Auriculariales (Auricu-
laria, Exidia), Cantharellales (Tulasnella, possibly
Ceratobasidium and other teleomorphs of Rhizoc-
tonia), and Sebacinales (Weiss et al. 2004; Binder
et al. 2005; Hibbett 2006; Moncalvo et al. 2006).
The class Tremellomycetes as currently circum-
scribed contains the well-delimited Tremellales
and two orders of yeasts, Cystofilobasidiales and
Filobasidiales. This placement of most if not all
agaricomycetous yeasts in the vicinity of Tremella
confirms earlier results (Fell et al. 2001) and cor-
relates with the germination of Tremella basidio-
spores as yeast cells.
Many phylogenetic studies have attempted to
subdivide fungi currently grouped in the class
Agaricomycetes (Hibbett et al. 2007), approxi-
mately equivalent to Homobasidiomycetes. Major
consternation arose when it became apparent just
how frequently the various forms of basidiocarps
and hymenophore – chiefly lamellate, cantharel-
loid, poroid, clavate, hydnoid, resupinate, and
gasteroid – appeared in different phylogenetic
groups (Fig. 1.6). Key biological features have also
evolved repeatedly. To give one example, ectomy-
corrhizal associations may have arisen indepen-
dently 11 times in the Agaricales (Matheny et al.
2006), twice in Boletales (Binder and Hibbett
2006), and on several occasions elsewhere within

Fig. 1.6. Distribution of hymenial surfaces among different orders of Agaricomycetes (Homobasidiomycetes).
Phylogenetically closely related orders are united by brackets
 Recent Developments in the Molecular Taxonomy of Fungi
Agaricomycetes (Hibbett and Thorn 2001). In the
Sebacinales, a small order of heterobasidiomycete-
like fungi now placed at the base of Agaricomycetes,
different types such as ectomycorrhiza, ericoid, and
orchid mycorrhiza may all be found (Weiss et al.
2004). Many groups therefore lack any obvious char-
acter by which their members can be recognised.
Initial attempts at re-classifying agaricomyce-
toid fungi defined phylogenetic clades instead of
formal order names (Hibbett et al. 1997; Hibbett
and Thorn 2001; Moncalvo et al. 2002; Webster and
Weber 2007). Orders have now been formally re-
described for the eight clades of Hibbett and Thorn
(2001), i.e. cantharelloid (Cantharellales; Moncalvo
et al. 2006), hymenochaetoid (Hymenochaetales;
Larsson et al. 2006), gomphoid-phalloid (Geas-
trales, Gomphales, Hysterangiales, Phallales; Hos-
aka et al. 2006), russuloid (Russulales; Miller et al.
2006), boletoid (Boletales; Binder and Hibbett
2006), euagarics (Agaricales; Matheny et al. 2006),
polyporoid (Polyporales; see Hibbett and Thorn
2001; Matheny et al. 2007), and thelephoroid (The-
lephorales; see Hibbett et al. 2007). Additional
(mostly smaller) clades were resolved subsequent
to the summary by Hibbett and Thorn (2001), and
these were described as Trechisporales, Sebacinales,
Gloeophyllales, Corticiales, and Atheliales (see
Weiss et al. 2004; Binder et al. 2005; Hibbett et al.
2007). However, a complete sub-class system is not
yet in place, only two groups (Agaricomycetidae
and Phallomycetidae) having been formalised in
the new taxonomic context as yet (Hibbett et al.
2007). Indeed, a new research discipline, fungal
phylogenomics, may have to be established before
the Agaricomycetes can be tidied up (Hibbett 2006),
and even then a working taxonomic system based
on well-known species may be destabilised by the
future discovery of new species from the immense
unknown biodiversity thought to exist among
Agaricomycotina.
VI. Conclusions
Modern multi-gene phylogenetic approaches are
carrying forward the revolution of fungal taxono-
my begun by single-gene studies some two decades
ago and are beginning to place the resulting tax-
onomy on a firmer, more ‘natural’ base. However,
as we have seen, many areas of ambiguity remain.
In addressing them, every effort should be made
to reduce name changes to the bare minimum, i.e.
13
to situations in which there is sound evidence of
their necessity, general acceptance, and future du-
rability. Further, a hierarchical scheme with a
limited number of phyla should be maintained
from the perspective of mycology teaching. Each
phylum should contain an equivalent structure of
lower ranks. In this context a re-erection of the
Zygomycota or an equivalent in rank is desirable.
The ‘AFTOL’ project has a model character in
addressing most of the above requirements, and it
is particularly promising in that so many influential
taxonomists have united behind it. It is certainly
possible to adopt this classification for use with
existing textbooks such as Webster and Weber
(2007), new texts or editions, or as a framework
for mycology classes. The AFTOL scheme presents
the first realistic opportunity to introduce a unified
taxonomic scheme into global mycological use.
However, taxonomy is merely a foundation on
which the ultimate goal of mycology – an under-
standing of the biology of fungi – can be
approached. In many institutions, the teaching of
mycology at undergraduate level is currently
being carried out by non-mycologists. The per-
ception of fungal taxonomy as an obsession with
name changes of ever shorter shelf-life must be
avoided, as this would simply lead to the elimina-
tion of fungi from biology textbooks and curri-
cula, as has arguably happened with protozoa
(Adl et al. 2007). In this context as in many others,
one must hope for a high acceptance and durabil-
ity of the AFTOL scheme.
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2 Sordaria macrospora, a Model System for Fungal Development

ULRICH KÜCK1, STEFANIE PÖGGELER2, MINOU NOWROUSIAN1, NICOLE NOLTING2,*, INES ENGH1

CONTENTS studies with model organisms have led, for

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 example, to the identification of signal transduction
A. Fungal Organisms as Model Systems pathways, transcription control circuits, and cell
for Developmental Biology . . . . . . . . . . . . . . . . . 17 cycle checkpoints. Moreover, studies using model
B. Why Choose Sordaria macrospora? . . . . . . . . 18 systems continue to uncover the details of genetic
II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
A. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 interactions controlling developmental plasticity.
B. Homothallism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 The acquired knowledge usually can be applied to
III. Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 organisms of higher order having more complex
IV. Mutants and Morphology . . . . . . . . . . . . . . . . . . . . . . . . 22 morphologies that are difficult to manipulate exper-
V. Molecular and Genetic Tools . . . . . . . . . . . . . . . . . . . . 24 imentally. A further advantage of model systems
A. Tetrad Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
B. DNA-Mediated Transformations compared to complex highly developed organisms
and Gene Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . 26 (e.g. humans) is their potential for a rapid and
C. Tools for Fluorescence Microscopy . . . . . . . . 27 inexpensive genetic analysis.
D. Functional Genomics . . . . . . . . . . . . . . . . . . . . . . . 30 In this review, we focus on the filamentous
VI. Developmental Biology and Components fungus Sordaria macrospora, which for long has
of Signalling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
A. Pheromones and Pheromone Receptors . . 31 been used as a model for conventional genetic anal-
B. Heterotrimetic G Proteins . . . . . . . . . . . . . . . . . . 31 ysis, fruiting body development, and the analysis of
C. Adenylyl Cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 meiosis (Esser and Straub 1958; Heslot 1958; Zickler
D. Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . 33 1977; van Heemst et al. 1999; Pöggeler et al. 2006a).
E. Novel Developmental Proteins . . . . . . . . . . . . . 34 Since the first combination of classical tetrad analy-
VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 sis and modern molecular genetics, this fungus has
become an important eukaryotic model system to
study developmental biology.
While this review was in preparation, the
genome of S. macrospora was sequenced, using
I. Introduction next generation sequencing techniques. De novo
assembly of Illumina/Solexa paired end reads and
The application of forward or reverse genetic subsequent comparative assembly with genome
approaches in model organisms has made great data from Neurospora species yielded a first draft
contributions to our fundamental biological knowl- version with about 10 000 predicted genes. This
edge. Associated with these studies is the technical genome sequence will greatly enhance the value
progress in understanding and manipulating the of S. macrospora as a eukaryotic model system
genomes of the relevant organisms. Basic biological (Nowrousian M, Kamerewerd J, Engh I, Pöggeler
S, Stajich J, Read N, Kück U, Freitag M, personal
1
Lehrstuhl für Allgemeine und Molekulare Botanik, Fakultät für communication).
Biologie, Ruhr-Universität Bochum, Universitätsstrasse 150,
44780 Bochum Germany; e-mail: ulrich.kueck@ruhr-uni-
bochum.de
2
A. Fungal Organisms as Model Systems
Abteilung Genetik eukaryotischer Mikroorganismen, Institut für
Mikrobiologie und Genetik, Georg-August-Universität Göttin-
for Developmental Biology
gen, Grisebachstrasse 8, 37077 Göttingen, Germany; e-mail:
spoegge@gwdg.de So far about 74 000 fungal species have been identi-
*
Present address: Heinrich Petk Institute for Experimental Virology fied and classified, but even conservative estimates
and Immunology at the University of Hambury, Germany suspect that about 1.5
106 species exist in nature

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
18 Ulrich Kück et al.
(Hawksworth 2001). Fungi are true eukaryotes,
with a nucleus and (in most cases) mitochondria.
Exceptions are those species having mitosomes
or mitochondria-related hydrogenosomes instead
of mitochondria. However, recent evolutionary
studies indicate that both mitosomes and hydroge-
nosomes might be derived from mitochondrial
ancestors (Embley and Martin 2006). Fungi can be
either uni- or multicellular. Irrespective of their
taxonomy, unicellular fungi are named yeasts,
while the terms filamentous or mycelial fungi indi-
cate multicelluar fungi with complex, multicellular
propagation structures. Depending on their envi-
ronmental growth conditions, some fungi are able
to undergo a transition from uni- to multicellular
growth and vice versa. This dimorphic develop-
ment is a characteristic of certain species that are
considered to be either yeasts or filamentous fungi.
Baker’s yeast, Saccharomyces cerevisiae, is the
most prominent fungal model system and has
contributed enormously to the basic understand-
ing for example of eukaryotic signalling pathways
or the biogenesis of eukaryotic organelles (e.g.
mitochondria, peroxisomes, endoplasmic reticu-
lum). Thus, yeast is the main model system for
eukaryotic cell biology (Botstein and Fink 1988).
However, multicellular models are essential
since many genes play different roles in defined
cell types at different developmental stages. These
complex cell and tissue interactions cannot be
mimicked in unicellular organisms like yeasts.
Therefore other model organisms, such as
filamentous fungi, have proven their value in
specialized research areas.
Filamentous fungi are characterized by the
extreme polar growth of their hyphae, which is
reminiscent of the growth behaviour in neurons or
plant pollen tubes. This polarized growth in fungi is
characterized by vesicle transport towards the apex.
In contrast to yeasts, filamentous fungi can undergo
major morphogenetic changes that are accompa-
nied by a switch from polarized to isotropic growth
(Harris and Momany 2004; Fischer et al. 2008).
These changes in cellular development occur regu-
larly during asexual or sexual propagation.
During asexual propagation, the typical for-
mation of conidiospores is observed (e.g. Asper-
gillus spp., Penicillium spp.). Another, often
overlooked, asexual differentiation process is the
fragmentation of hyphae into arthrospores. Both
these processes are dependent on environmental
conditions, such as light.
Similarly, sexual propagation structures occur
in true filamentous fungi due to environmental
changes such as nutrient starvation. In almost all
filamentous fungi, fruiting body formation is the
major developmental step during the sexual
reproduction cycle. Fruiting bodies vary in size
between 200 mm and several centimetres as found
for example in the morel. Their major function is
the protection and further distribution of the sexu-
al spores that are generated after the meiotic divi-
sions that follow karyogamy (Pöggeler et al. 2006a).
B. Why Choose Sordaria macrospora?
Sordaria macrospora is a coprophilic homothallic
pyrenomycete first described in 1866 (Auerswald
1866), which is closely related to Podospora anser-
ina and Neurospora crassa. In contrast to these
heterothallic species, single strains of S. macro-
spora produce fruiting bodies (perithecia) without
the presence of a mating partner. As a conse-
quence of self-fertility, fruiting body development
is an apandrous process, allowing the direct test-
ing of recessive mutations that lead to defects in
fruiting body formation. The life cycle of S. macro-
spora, as depicted in Fig. 2.1, can be completed in
the laboratory within seven days. During vegeta-
tive growth, chemical and physical stimuli such as
biotin or light induce branching of hyphal tips,
which is followed by adhesion of several hyphae to
each other. This entry into the sexual phase results
in the formation of a network of interconnecting
hyphae, leading to the first of two consecutive
morphological stages: first, the development of
primordia (protoperithecia), and second, the
transition of protoperithecia into mature fruiting
bodies, called perithecia. In the homothallic
fungus S. macrospora, transition between these
two stages takes 72 h. In heterothallic fungi such
as N. crassa, this transition requires crossing
between two strains of opposite mating types.
Ascus development, as detailed in the next
section, starts with the formation of female game-
tangia called ascogonia. The ascogenous cells are
enveloped by sterile hyphae to form fruiting body
precursors. Subsequent tissue differentiation
gives rise to an outer pigmented peridial tissue
and, following karyogamy, inner ascus initials
embedded in sterile paraphyses are formed.
Mature fruiting bodies from S. macrospora harbor
50–300 asci, which after meiosis and postmeiotic
 Sordaria macrospora, a Model System for Fungal Development
ascospore
perithecium with
mature asci mycelium
ascogonium
protoperithecium
perithecium with
immature asci
divisions contain eight ascospores each. Mature
black ascospores are discharged through an
apical pore (ostiole) at the neck of the fruiting
body. In conclusion, fruiting body development
requires the differentiation of the mycelia into
several specialized tissues; and regulation of
these morphological and physiological changes
requires a number of different genes (Esser and
Straub 1958).
There is another developmental feature that
makes S. macrospora a favourable object for fruit-
ing body development. S. macrospora produces
only meiotically derived ascospores, whereas
asexual spores, such as conidia, are absent. Thus,
there is no interference between two different
developmental programmes, which makes it easi-
er, for example, to analyse differentially expressed
genes involved in ascocarp development.
II. Biology
In order to manipulate the development of a fungal
model system, detailed knowledge of its life cycle
is an essential prerequisite for any experimental
study. The sexual system of fungi can be either het-
erothallic (self-incompatible), pseudo-homothallic
19
Fig. 2.1. Life cycle of Sordaria
macrospora: The cycle starts
with a germinating ascospore
and is completed in the labora-
tory within seven days by the
formation of mature fruiting
bodies (perithecia) that dis-
charge the mature and mela-
nized ascospores
(secondary homothallic, pseudo-compatible), or
homothallic (compatible) (Lin and Heitman 2007).
A. Life Cycle
Similar to heterothallic filamentous ascomycetes,
the homothallic S. macrospora has a dikaryophase
in its life cycle. During this phase, pairs of nuclei
synchronously and repeatedly divide inside the
ascogonium, thereby producing a great number
of nuclei. The nuclei then migrate in pairs to
the developing dikaryotic ascogenous hyphae
emerging from the ascogonium. The ascogenous
hyphal tips develop a U-shaped crozier containing
two nuclei. These nuclei divide mitotically in
synchrony and, subsequently, two septa appear
which divide the hook cell into three sections: a
lateral and a basal cell with one nucleus each, and
an apical cell with two nuclei. The two nuclei in the
apical cell then fuse. Immediately after karyogamy
within the young ascus, the diploid nucleus under-
goes meiosis, which is followed by a post-meiotic
mitosis, resulting in the formation of eight nuclei.
Each of the eight nuclei is incorporated into its
own ascospore (Esser and Straub 1958; Esser
1982). The self-fertilization mode of S. macrospora
is termed autogamous fertilization. This means
20
that a pair-wise fusion of nuclei present within the
ascus initial occurs, without cell fusion having
taken place before (Esser and Straub 1958).
B. Homothallism
In homothallic species, like S. macrospora, a
mycelium derived from a uninucleate ascospore
is self-fertile and able to perform all steps of
meiosis. In contrast, heterothallic species, like
N. crassa, require another compatible individual
for sexual reproduction. The mating partners of
heterothallic fungi are morphologically identical
and are only distinguished by their mating type
(MAT) loci. In N. crassa, these are termed MAT1-1
(matA) and MAT1-2 (mata). The alternative ver-
sions of the mating-type locus on homologous
chromosomes of the mating partners are called
idiomorphs because they are completely dissimi-
lar in the genes they carry (Metzenberg and Glass
1990). The MAT1-1 idiomorph of N. crassa con-
tains three genes: MAT1-1-1 (mat A-1), MAT1-1-2
(mat A-2), and MAT1-1-3 (mat A-3; Glass et al.
1990a; Ferreira et al. 1996, 1998; Fig. 2.2). The
MAT1-1 polypeptide has a DNA-binding motif
that shows similarity to the S. cerevisiae MATa1p
mating-type protein. MAT1-1-2 encodes a protein
without a characteristic DNA-binding motif, but
with a conserved region encompassing 25 amino
acids with three invariant histidine, proline, and
glycine residues (the HPG domain; Debuchy
and Turgeon 2006). The MAT1-1-3 gene encodes
a protein with a high mobility group (HMG)
DNA-binding motif (Ferreira et al. 1996). The
N. crassa MAT1-2 idiomorph contains two genes,
MAT1-2-1
(mat a-1)
N.c. MAT1-2
HMG
N.c. MAT1-1
MAT1-2-1
(Smta-1)
S.m. MAT1-2/1
HMG
Fig. 2.2. Comparative maps of mating type loci from the two N. crassa (N. c.) mating type idiomorphs and from the
S. macrospora (S. m.) mating type locus. For further details of designations see text
Ulrich Kück et al.
MAT1-2-1 (mat a-1) and MAT1-2-3 (mat a-2).
While the MAT1-2-1 gene encodes a HMG-do-
main protein that is the major regulator of mating
in MAT1-2 strains, the function of the MAT1-2-2
encoded protein is unknown (Staben and
Yanofsky 1990; Pöggeler and Kück 2000).
Pseudo-homothallic members of the Sordar-
iaceae (e.g. Neurospora tetrasperma) or the
Lasiosphaeriaceae (e.g. P. anserina) develop
four-spored asci in which most ascospores carry
two nuclei, one of each mating type. Another form
of pseudo-homothallism is accomplished by mat-
ing-type switching, but this has not been identi-
fied in members of the Sordariales (Lin and
Heitman 2007).
There are three ways fungi can be homothal-
lic: (1) they can harbour a fused MAT locus of
both idiomorphs, (2) they can harbour both MAT
alleles at different loci in the genome, and (3) they
can sexually reproduce but carry only one MAT
locus (Lin and Heitman 2007). In Sordariales all
three forms of homothallism can be found in the
same order.
S. macrospora as well as Neurospora pannonica and Neu-
rospora terricola belong to the first group and contain
sequences similar to both the MAT1-1 and the MAT1-
2 idiomorphs. In the homothallic Chaetomium globosum
in addition to a MAT1-1 locus, a MAT1-2 locus was iden-
tified at another genomic locus (Glass et al. 1990a; Pögge-
ler et al. 1997a; Debuchy and Turgeon 2006). However,
according to data from Southern hybridization, the homo-
thallic Neurospora species N. africana, N. dodgei, N. gala-
pagonensis, and N. lineolata possess only the MAT1-1 locus
(Glass et al. 1990a; Pöggeler 1999). The fused mating-type-
loci of S. macrospora, N. pannonica, and N. terricola appear
to be derived from a heterothallic ancestor. The molecular
mechanisms responsible for the fusion of MAT regions are
MAT1-2-1
(mat a-2)
?
MAT1-1-3 MAT1-1-2 MAT1-1-1
(mat A-3) (mat A-2) (mat A-1)
HMG HPG a
MAT1-1-3 MAT1-1-2 MAT1-1-1
(SmtA-3) (SmtA-2) (SmtA-1)
? HPG a
 Sordaria macrospora, a Model System for Fungal Development
so far unknown, but it is likely that a recombination event
led to the close linkage of MAT1-1- and MAT1-2-specific
sequences on a single chromosome.
Cloning and sequencing of the entire mating-type
locus of S. macrospora revealed that it encodes four
different ORFs: MAT1-2-1 (Smt a-1), MAT1-1-3
(Smt A-3), MAT1-1-2 (Smt A-2), and MAT1-1-1
(SmtA-1). Except for MAT1-1-3, proteins encoded
by the S. macrospora mating type genes show
strong sequence similarities to the corresponding
N. crassa mating-type ORFs. The S. macrospora
MAT1-1-3 gene has a chimeric character and exhi-
bits sequence similarity to the MAT1-1-3 and the
MAT1-2-2 ORF of N. crassa. In contrast to the N.
crassa MAT1-1-3, the S. macrospora MAT1-1-3
protein lacks an HMG domain (Fig. 2.2).
Sequence analysis of the cDNA from the S.
macrospora MAT1-2-1, MAT1-1-3, MAT1-1-2,
and MAT1-1-1 revealed that introns in each gene
are spliced, indicating that all of the mating-type
genes identified in the mating-type locus of
S. macrospora are transcriptionally active (Pögge-
ler et al. 1997b). Surprisingly, RT-PCR experi-
ments demonstrated co-transcription of the
MAT1-1-3 gene and MAT1-2-1 as well as optional
splicing of two introns within the MAT1-1-2 gene
(Pöggeler and Kück 2000).
To investigate the functional conservation of
mating-type genes of S. macrospora, cosmid
clones containing the entire mating-type locus
from S. macrospora were transformed into both
P. anserina MAT1-1 and MAT1-2 strains. Similar
to N. crassa MAT1-1 strains, the P. anserina
MAT1-1 contains three genes (FMR1 = MAT1-1-
1, FMR2 = MAT1-1-2, SMR3 = Mat1-1-3), while
the P. anserina MAT1-2 locus contains only a
single gene (FPR1 = MAT1-2-1) encoding an
HMG-domain protein (Debuchy and Coppin
1992; Debuchy et al. 1993). After introduction of
the S. macrospora MAT locus into the homokar-
yotic strains of P. anserina, transformants were
capable of forming fruiting bodies without cross-
ing with a mating partner of the opposite mating
type. However, instead of asci with four ascos-
pores, the perithecia of the transformants contain
only a gelatinous mass and have no structures
such as hook cells, croziers, asci or spores
(Pöggeler et al. 1997b). Interestingly, transforma-
tion of the entire S. macrospora MAT locus into a
P. anserina DMAT strain without an endogenous
MAT locus led to fertile perithecia containing
21
rosettes of asci (Pöggeler et al. 1997b). Thus, the
mating-type genes from S. macrospora confer self-
fertility to the heterothallic P. anserina when they
do not interfere with a resident mating-type locus.
This result suggests that the S. macrospora mat-
ing-type genes are functional and most probably
are involved in fruiting body development and
ascosporogenesis.
Due to the presence of MAT1-1-type homo-
thallic species in the genus Neurospora (Glass
et al. 1990b; Metzenberg and Glass 1990) and due
to the fact that MAT1-1 strains of the heterothallic
S. brevicollis were shown to produce apandrous
fruiting bodies with ascospores under certain
culture conditions (Robertson et al. 1998), it was
unclear whether MAT1-2 specific genes are essen-
tial for sexual reproduction in homothallic mem-
bers of the family Sordariaceae. Deletion of
MAT1-2-1 converts the self-fertile S. macrospora
to a self-sterile fungus, no longer able to produce
fruiting bodies and ascospores. Thus, the DMAT1-
2-1 mutant phenotypically resembles the
S. macrospora pro mutants (see Sect. IV) which
also do not produce any perithecia. However, no
irregularities in vegetative growth and mycelial
morphology were observed when the mutant
strain was compared to S. macrospora wild type
This finding demonstrates that, at least in the
homothallic S. macrospora, the MAT1-2-1 gene
is required for fruiting-body development and
sexual reproduction (Pöggeler et al. 2006b).
Cross-species microarray technology (see
Sect. V.D) showed that the S. macrospora MAT1-
2-1 affects the expression of at least 107 genes,
including a common set of ten putative develop-
mental genes deregulated in sterile pro mutants.
At least 74 and 33 genes are two-fold up- or down-
regulated in the S. macrospora DMAT1-2-1 mutant
strain. Of these 107 genes, 80 have homologues
with known or putative function and were sorted
into ten putative functional categories (Pöggeler
et al. 2006b).
Transcription of cell type-specific genes in the
ascomycetous yeast Saccharomyces cerevisiae has
been shown to rely on the interaction of mating-
type proteins (Herskowitz 1989). A two-hybrid ap-
proach in conjunction with protein cross-linking
analysis demonstrated that in Sordaria macrospora
the MAT1-1-1 and MAT1-2-1 proteins, homolo-
gous to polypeptides encoded by opposite mating
partners of the heterothallic N. crassa, are able to
form a heterodimer (Jacobsen et al. 2002).
22 Ulrich Kück et al.
III. Phylogeny
S. macrospora belongs to the family Sordariaceae
(Sordariales, Ascomycetes) which comprises taxa
characterized by dark, ostiolate fruiting bodies, and
unitunicate, cylindrical asci (Kirk et al. 2001). The
Sordariaceae family presently contains 7–10 genera
(Kirk et al. 2001, Eriksson et al. 2004) and is
represented by well known genera such as Gelasi-
nospora, Neurospora, and Sordaria. It is also mor-
phologically related to the Lasiosphaeriaceae,
another family within the Sordariales, which con-
tains the important genus Podospora (Huhndorf
et al. 2004). Phylogenetic studies have revealed
that species of the genus Sordaria are closely
related to the genus Neurospora (Pöggeler 1999;
Cai et al. 2006) which includes the fungal model
organism N. crassa (Davis and Perkins 2002; Gala-
gan et al. 2003; Borkovich et al. 2004; Fig. 2.3).
Phylogenetic trees based on sequences of the
conserved glyceraldehyde-3-phosphate dehydro-
genase gene (gpd) and on gene fragments
of MAT1-1-1 and MAT1-2-1 have shown that
Neurospora and Sordaria are monophyletic units
(Pöggeler 1999). Moreover, there is a strict sepa-
ration of heterothallic and homothallic species
within both genera and a separation of homothal-
lic strains with fused mating-type loci and MAT1-
1-type homothallic species within Neurospora.
The phylogenetic analyses suggest that changes
in the reproductive strategy may represent a
single event in each genus (Pöggeler 1999).
The MAT genes of members of the Sordaria-
ceae appear to evolve more quickly than other
regions of the genome; a phenomenon also
observed in other ascomycetes (Turgeon 1998;
Pöggeler 1999; Voigt et al. 2005). Interestingly,
analysis of non-synonymous and synonymous sub-
stitution rates within MAT genes of the genus Neu-
rospora revealed that the rapid divergence of MAT
genes is due to an adaptive evolution within het-
erothallic members of this genus, whereas it is due
to the lack of selective constraints within homo-
thallic members of Neurospora (Wik et al. 2008).
IV. Mutants and Morphology
A major step in the molecular genetic analysis of
components controlling cellular development is
the functional analysis of mutants having a defect
or block in defined morphogenetic steps. Such
morphological mutants can be generated either
by conventional mutagenesis or by the application
of homologous recombination procedures (see
Sect. V.B). The detailed molecular and cellular
analysis of mutants will decipher molecular deter-
minants involved in cellular differentiation pro-
cesses. Using a conventional genetic approach,
more than hundred developmental mutants with
defects in fruiting body formation were generated.
Mutants were obtained after UV mutagenesis of a
protoplast suspension derived from the wild-type
strain. Protoplasts were exposed to UV light (254
nm) for 15 min, with a survival rate of 0.1%.
The protoplasts were regenerated on complete
medium, supplemented with 10.8% sucrose
(Masloff et al. 1999). After 24 h, individual clones
were transferred to corn-meal medium to investi-
gate clones with phenotypic variations in fruiting
body development. In total, more than 23 000
clones were screened in this way. Presumptive
developmental mutants were further tested for
mitotic stability, before single-spore isolates were
generated for a detailed molecular analysis.
The mutants were grouped into four different
phenotypic classes (see Fig. 2.4A):
1. Asc mutants. These mutants show a very early
developmental block in the life cycle. They form
ascogonia that sometimes have a non-wild-type
structure. In more than 100 developmental
mutants, we detected only five having this phe-
notype. They are sterile and never form proto-
perithecia.
2. Pro-mutants (Fig. 2.4B). Some 45 mutants have
been isolated and they show a developmental
block just after protoperithecia formation.
Therefore, all pro-mutants are sterile. Proto-
perithecia have a round-shaped structure and
a diameter of 30–55 m. The average size can
vary in the different mutants but has been
found to be constant in a defined mutant when
grown on corn meal agar. As shown in Sect. VI.
E, these mutants have contributed greatly to our
current understanding of the molecular genetic
network controlling fruiting body development.
The first mutant used for a complementation
analysis was pro1, where the pro1 gene encodes
a development-specific GAL4-like C2H6 tran-
scription factor (Masloff et al. 1999).
3. Per-mutants. The 44 mutants of this type are
characterized by the presence of a more or less
 Sordaria macrospora, a Model System for Fungal Development 23

N. tetrasperma#
92
+, heterothallic
#, pseudohomothallic 82 N. intermedia+
*, homothallic
?, uncertain mating system N. sitophila+
98
N. crassa+

N. discreta+

62 G. cerealis*
69

N. pannonica*

Sordariaceae
N. terricola*
91
N. lineolata*
84

N. africana*

G. tetrasperma#
100

S. macrospora*
100

100 S. lappae?

75 S. fimicola*

S. sclerogenia+
95

S. brevicollis+

Laiosphaeriaceae
P. anserina#

100 P. setosa*

C. coprophila?

Chaetomium globosum*
(outgroup)

Fig. 2.3. Phylogenetic tree of partial gpd sequences gener-
ated by neighbor-joining analysis. 436 nucleotide posi-
tions were included in the analysis. The tree shown is
based on a consensus tree calculated with the Neighbor
program (Phylip). The numbers (percentages) indicate the
bootstrap support based on 1000 replications. Abbrevia-
tions and accession numbers are as follows: N. tetrasperma
(Neurospora tetrasperma, AJ133018.1); N. intermedia (Neu-
rospora intermedia, AJ133019.1); N. sitophila (Neurospora
sitophila, AJ133020.1); N. crassa (Neurospora crassa,
U67457.1); N. discreta (Neurospora discreta, AJ133021.1);
G. cerealis (Gelasinospora cerealis, AF388934.1); G. tetra-
sperma (Gelasinospora tetrasperma, AF388935.1); N. pan-
nonica-. (Neurospora pannonica, AJ133016.1); N. terricola
(Neurospora terricola, AJ133017.1); N. lineolata (Neurospo-
ra lineolata, AJ133015.1); N. africana (Neurospora africana,
AJ133012.1); S. macrospora (Sordaria macrospora,
AJ133007.1); S. lappae (Sordaria lappae, EF197111.1);
S. fimicola (Sordaria fimicola, AJ133009.1); S. sclerogenia
(Sordaria sclerogenia, AJ133011.1); S. brevicollis (Sordaria
brevicollis, AJ133010.1); P. anserina (Podospora anserina,
EF197096.1); P. setosa (Podospora setosa, EF197110.1);
C. coprophila (Cercophora coprophila, EF197091.1). Chaeto-
mium globosum- (CHGG_07980.1; http://www.broad.mit.
edu/annotation/genome/chaetomium_globosum) was used
as the outgroup

developed perithecium, but otherwise they are
sterile. This is due to the fact that they
are unable to generate mature ascospores,
although in some cases, they are able to form
immature asci. A good example is mutant
per5 (see Sect. VI.E), which was one of two
mutants that provided the first example for
the applicability of the S. macrospora system
for a molecular genetic analysis (Nowrousian
et al. 1999).
24 Ulrich Kück et al.

200 µm 20 µm

p ile - m u tan ts asc - m u tan ts

16 5
16 5

45
44
45

100 µm 20 µm
-
p er - m u tan ts p ro - m u tan ts
A

20 µm 200 µm

B C

Fig. 2.4. Phenotypes of developmental mutants from Sordaria macrospora. A Life cycle and number of mutants that can
be grouped in four different morphological classes having distinct developmental blocks. B, C Scanning electron
micrographs of a pro (B) and pile mutant (C)

4. Pile mutants (Fig. 2.4C). These mutants show a
rather complex phenotype. The spatial order of
perithecium formation is disturbed. One peri-
thecium can sit on top of another, thus forming
a pile of many perithecia. Initially, we obtained
16 isolates of this mutant type which seem to
be genetically instable. Pile mutants are often
fertile, but show a defect in the formation of the
pigment melanin (Engh et al. 2007a).
As described in the sections to come, differ-
ent developmental mutants were used for the fur-
ther molecular investigation of the developmental
process. Restoration of a fertile phenotype in these
sterile mutants allowed isolation of a wild-type
gene copy, which is defective in the recipient
strains.
V. Molecular and Genetic Tools
Development of an organism as a genetic model
system is greatly dependent on the application of
modern molecular genetic techniques to a hitherto
only conventionally manipulated genetic system. In
S. macrospora the use of molecular genetics started
with DNA-mediated transformations and recently
applied techniques such as microarray hybridiza-
tion and novel cellular markers for fluorescent
 Sordaria macrospora, a Model System for Fungal Development 25

microscopic investigations (Nowrousian et al. 2005;

Engh et al. 2007b; Nowrousian et al. 2007a).

A. Tetrad Analysis
Tetrad analysis investigates the four products that
are generated from a diploid cell during two meiotic
divisions. In fungi, meiosis occurs during sexual
spore formation, resulting in each of the spores in
the meiosporangium (the ascus in the case of asco-
mycetes) resembling one product of meiosis.
In ascomycetes ordered tetrads as can be found in
S. macrospora can be distinguished from non-
ordered tetrads present in yeast. If alleles are
segregated during the first meiotic division, this
leads to ascus halves in ordered tetrads with spores
of the same genotype. However, cross-over may
occur during prophase of the first meiotic division
between the centromere and the gene of interest.
Different alleles are then present on sister chroma-
tids and do not segregate until the second meiotic
division. This leads to ascus halves with spores of
different genotypes. For further analysis, spores can
be isolated and germinated on agar medium.
S. macrospora is an excellent organism for
tetrad analysis for several reasons (Esser
Fig. 2.5. Ascus rosettes from a cross between two different
and Straub 1958; Esser 1982), some of which are S. macrospora strains. A Tetrad analysis from a cross
outlined here. between a wild-type strain having black ascospores and a
The tetrad is ordered and the ascospores are mutant strain with yellow ascospores. Light (B) and fluo-
linearly arranged. A post-meiotic mitosis leads to rescence (C) micrographs from ascus rosettes that were
eight instead of four spores. Sister products derived from a cross between a strain carrying two differ-
ent ascospore colour mutations and a transformant carry-
remain adjacent to one another; thus, the position ing the gene for the green fluorescence protein (gfp).
of each ascospore reflects the preceding nuclear Arrows indicate asci in which the alleles for the ascospore
division. colours or green fluorescent protein were separated by
The ascospores of S. macrospora are about 28 first and second division segregation, respectively
mm long, allowing for easy manual isolation.
Several spore colour mutants exist that can What is the advantage of a tetrad analysis?
be used in crosses. This is important, because Traditionally, it is used to examine gene distribu-
S. macrospora is self-fertile and one cannot distin- tion and gene–centromere distances by analysing
guish selfed and non-selfed fruiting bodies. the frequency of second division segregation. This
However, when using a strain with a spore colour was made mostly obsolete by genome sequencing.
defect in sexual crosses, recombinant perithecia However, tetrad analysis is still a powerful tool for
are identified by the segregation of spore colour genetic analysis. For example, recently a Dku70
alleles within the asci (Fig. 2.5). mutant was generated that facilitates the genera-
Experiments have shown that not only spore tion of knockout strains (Pöggeler and Kück
colour alleles but also heterologous marker genes 2006), which still carry the ku70 deletion (see
like egfp segregate in a Mendelian manner (Pöggeler next chapter). In order to obtain a knockout strain
et al. 2003). in the wild-type background, crosses against a
S. macrospora has a very short sexual cycle spore colour mutant are performed for further
that can be completed within seven days under tetrad analysis. This is a fast and easy method
laboratory conditions. leading to homokaryotic mutant strains
26 Ulrich Kück et al.
(Pöggeler and Kück 2006). Another important ad-
vantage of a tetrad analysis is the possibility to
generate double mutants by crossing the two
mutants carrying distinct single mutations. The
double mutant can be isolated even if: (1) both
single mutants were generated using the same
selection marker, or (2) the double mutant has
the same phenotype as one of the single mutants.
This is feasible, because segregation into parental
and recombinant spores is taken into account and
the ordered tetrad allows for the identification of
both. Using this method, numerous S. macrospora
double mutants have been generated (Mayrhofer
et al. 2006; Kamerewerd et al. 2008). As a conse-
quence, conventional genetic analysis strongly
increases the potential of S. macrospora as a
model organism for studying diverse biological
phenomena.
B. DNA-Mediated Transformations
and Gene Libraries
A variety of options recently became available to
introduce and control genetic elements, as well as
to help the construction of different molecular
libraries, displaying the importance of S. macro-
spora as model organism for basic research (Kück
and Pöggeler 2005).
At the beginning, the development of a repro-
ducible DNA-mediated transformation protocol
was of vital importance. Transformation of
S. macrospora was first reported by Le Chevanton
and co-workers. They used an auxotrophic ura5
strain as the recipient. Appropriate selection
was performed by complementation of the uracil
auxotrophy, by transforming with a homologous
ura5 gene (Le Chevanton et al. 1989).
Selection of S. macrospora transformants with
a dominant marker became feasible using the
Escherichia coli hygromycin B phosphotransferase
gene (hph) under control of fungal promoter and
terminator elements (Walz and Kück 1995).
The advantage of such a dominant selectable
marker is that basically any recipient strain can
be used in transformation experiments. Expres-
sion of the hph gene was initially directed by the
upstream region of the isopenicillin N synthetase
gene (pcbC) from the cephalosporin C producing
fungus Acremonium chrysogenum. Recently, the
strong constitutive promoters of the A. nidulans
gpd and trpC genes were shown to be useful for
driving hph expression (Walz and Kück 1995;
Pöggeler et al. 2003), with transformation fre-
quencies of about 5–15 transformants per 10 mg
of DNA (Walz and Kück 1995).
In order to generate an alternative dominant
marker system that does not exhibit cross-resis-
tance to hygromycin B, the nat1 gene conferring
resistance to nourseothricin was used to trans-
form S. macrospora (Hoff and Kück 2006). The
nat1 gene product is the nourseothricin acetyl-
transferase from Streptomyces noursei (Krügel
et al. 1993) and was expressed under the control
of the A. nidulans trpC promoter. Transformation
frequencies of 10–40 transformants per 10 mg of
plasmid DNA were obtained with this dominant
selection system (Hoff and Kück 2006).
Alternative selection systems were developed
based on novel auxotrophic recipient strains.
Using conventional mutagenesis, a leucine auxo-
trophic strain was generated, which lacks the com-
plete wild-type copy of the leu1 gene, encoding
b-isopropylmalate dehydrogenase (Kück 2005). A
uracil auxotrophic mutant strain was constructed
by isolating the ura3 (syn. pyr4) gene, encoding
orotidine-50 -phosphate decarboxylase from the S.
macrospora genomic DNA (Nowrousian and Kück
1998) and subsequently disrupting the ura3 open
reading frame in vitro by insertion of a hph resis-
tance cassette. The resulting recombinant plasmid
was used in S. macrospora transformations to iso-
late a fungal derivative, in which the wild-type ura3
copy was substituted by the chimeric ura3-hph
gene through site specific homologous recombina-
tion (Kück and Pöggeler 2004). The leu1 and ura3
strains can be transformed with the corresponding
wild-type genes from S. macrospora; and the trans-
formation frequency is comparable to that
described for the hph gene.
Transformed DNA was shown to be ectopically
integrated into the genomic DNA of S. macrospora.
The copy number varied between transformants.
Interestingly, the analysis of S. macrospora trans-
formants by pulse field gelelectrophoresis revealed
that all vector molecules usually integrate into the
same chromosome (Walz and Kück 1995).
Targeted gene replacement via homologous re-
combination is a routinely used approach to
elucidate the function of unknown genes. Integra-
tion of exogenous DNA in the genomic DNA
requires the action of double-strand repair mechan-
isms. However, similar to most filamentous
fungi, plants, and animals, transformed DNA is
 Sordaria macrospora, a Model System for Fungal Development
predominately ectopically integrated into the
genome of S. macrospora.
Most probably this occurs by non-homologous end join-
ing (NHEJ), a mechanism that involves the binding of the
Ku heterodimer (Ku70/Ku80) at the ends of a DNA dou-
ble-strand break (DSB; Pastwa and Blasiak 2003).
Hence, the low rate of homologous recombination
exhibited by the S. macrospora wild-type has
hindered highly efficient gene knockout protocols.
In N. crassa, Ninomiya and co-workers
demonstrated that deletion of the genes ku70
and ku80 results in a dramatic increase in the
frequency of homologous recombination resulting
in 100% of transformants exhibiting integration
only at homologous sites (Ninomiya et al. 2004).
This strategy for targeted gene integration was
extended to S. macrospora and a ku70 deletion
mutant was generated by replacing the ku70 gene
with a nat1 resistance cassette. No phenotypic
defects regarding vegetative or sexual develop-
ment were observed in the ku70 strain, making it
an attractive recipient strain for transformation
and a valuable tool for gene disruption of devel-
opmental genes. Compared to the wild type, which
shows homologous integration efficiency between
0.1% and 5.0%, a Dku70 strain showed up to 100%
of transformants with homologous integration
of exogenous DNA (Pöggeler and Kück 2006).
As described in the previous section, the Dku70::
nat1 mutation can easily be eliminated from a
disruption strain by crossing to a spore color
mutant with an otherwise wild-type genetic back-
ground. Tetrad analyses allow the isolation of
ascospore isolates that lack the Dku70 mutation,
but carry the substituted gene copy.
Based on procedures described for N. crassa
(Colot et al. 2006), the S. macrospora transforma-
tion procedure was expanded by substituting con-
ventional cloning with recombinational cloning in
yeast (Oldenburg et al. 1997; Raymond et al. 1999).
Using this method, traditional restriction diges-
tion and ligation for the generation of knockout
fragments are unnecessary and components of the
final construct are synthesized individually by
PCR. Amplified fragments contain short overlap-
ping sequences and are cotransformed into
yeast for assembly by the recombination machin-
ery. This ‘‘two-step’’ transformation procedure
including homologous recombination in yeast
and transformation of the knockout fragment into
27
S. macrospora Dku70 strain presents a valuable tool
for gene disruption and a crucial improvement
for the molecular research on S. macrospora (Mayr-
hofer et al. 2006; Nolting and Pöggeler 2006a).
Genetic transformation protocols contribute
greatly to the molecular dissection of the S. macro-
spora sexual development. Another important
development for genetic engineering of S. macro-
spora comes from the construction of various
gene libraries of S. macrospora DNA. An indexed
cosmid library and a cDNA library, which is suit-
able for yeast two-hybrid screens, were used to
identify important components involved in sexual
reproduction and fruiting-body development
(Pöggeler et al. 1997a; Mayrhofer and Pöggeler
2005). Clones of indexed cosmid libraries are
kept individually in microtitre dishes to ensure
equal representation of each clone. Several meth-
ods for the rapid screening of pooled cosmid DNA
have been published, such as isolation of con-
served fungal genes by means of colony filter
hybridization of cosmid clones or rapid screening
by means of PCR using pooled cosmid DNA (Pög-
geler et al. 1997a). In other approaches, this
library was used to clone novel developmental
genes by restoring the fertile wild-type pheno-
types in otherwise sterile mutants of S. macro-
spora (see Sects. IV, VI.E; Masloff et al. 1999;
Nowrousian et al. 1999, 2007a; Pöggeler and Kück
2004; Engh et al. 2007b). The second genomic
library was generated for yeast two-hybrid studies
and consists of yeast vectors containing S. macro-
spora cDNA (Nolting and Pöggeler 2006a). This
library facilitated the screening of interaction part-
ners of developmental proteins and was shown to
be useful for the identification of transcriptional
activators and other developmental genes (Nolting
and Pöggeler 2006a).
C. Tools for Fluorescence Microscopy
Fluorescence microscopy has become a powerful
tool for studying the biology of filamentous fungi
in detail, e.g. gene regulation, signal transduction,
and protein localization. It relies on the fluores-
cent labelling of proteins or cellular components.
This can be achieved by different methods, i.e.
fluorescent dyes, fluorescent proteins or a combi-
nation of specific primary antibodies with conju-
gated secondary antibodies. While fluorescent
dyes are mostly used to label cell organelles or
28 Ulrich Kück et al.

membranes, fluorescent proteins and antibodies
are often employed to visualize proteins. All three
applications were conducted with S. macrospora
as an experimental system.
Using immunofluorescence, S. macrospora was
employed as a model organism for cytoskeleton
interactions during ascus development (Thomp-
son-Coffe and Zickler 1992, 1993), using antibodies
against actin and tubulins in combination with
secondary antibodies conjugated with fluorescent
probes. In addition, antibodies against meiotic pro-
teins like e.g. RAD-51 were generated for the
analysis of meiosis (Tessé et al. 2003; Storlazzi
et al. 2008). A similar approach was used to deter-
mine the subcellular localization of the develop-
mental protein ACL1 (Nowrousian et al. 1999).
The advantage of fluorescent proteins over
immunofluorescence is that no antibodies are
necessary and in vivo time-lapse imaging is possi-
ble. For studying the localization of developmen-
tal proteins, a number of plasmids with genes for
different fluorescent proteins were generated
(Table 2.1, Fig. 2.6). These plasmids encode
N- or C-terminal fusion proteins between the
gene of interest and the fluorescent protein.
These fluorescent markers segregate during mei-
otic division (Pöggeler et al. 2003). Using EGFP
and DsRed, the localization of the developmental
proteins PRO40, PRO41, and MCM1 were identi-
fied as Woronin bodies, the ER, and the nucleus,
respectively (Nolting and Pöggeler 2006b; Engh
et al. 2007b; Nowrousian et al. 2007a). For PPG1,
the pheromone precursor protein, and PRO41, a
novel component of the ER, putative signal
sequences for translational insertion into the ER
were verified by Western analysis with an anti-
EGFP-antibody that detected the secreted PPG1-
EGFP and PRO41-EGFP fusion proteins (Mayrho-
fer and Pöggeler 2005; Nowrousian et al. 2007a).
However, fluorescent proteins are not only
powerful tools for characterizing protein localiza-
tion. In fungi, they have been used to study the
dynamics of organelles (Mouriño-Pérez et al.
2006) and to screen for targeting mutants
(Ohneda et al. 2005). For S. macrospora, several
plasmids encoding organelle-targeted fluorescent
proteins were constructed (Table 2.1, Fig. 6A, C,
E–H) by fusing targeting signals to genes encod-
ing fluorescent proteins. One example is plasmid
pDsRed-SKL, encoding the DsRed gene fused to a
short sequence, encoding the SKL motif. This C-
terminal peroxisomal targeting sequence (PTS1)
targets the fusion protein to the peroxisome
(Elleuche and Pöggeler 2008). Likewise, ER mar-
kers pEGFP-KDEL and pDsRed-KDEL were con-
structed using EGFP and DsRed together with
the C-terminal retention signal KDEL and the N-
terminal secretion signal sequence of the ppg1
pheromone precursor gene (Mayrhofer and Pög-
geler 2005; Nowrousian et al. 2007a). The ER
marker proteins were successfully used to identify
the localization of fluorescent signals from
labelled developmental proteins (Engh et al.
2007b, Nowrousian et al. 2007a). Using organ-
elle-targeted GFP proteins, we have shown in two
mutants (pro40, pro41) that the Woronin body

Table 2.1. Plasmids encoding fluorescent proteins for microscopic investigations. Plasmids encode either solely the
fluorescent protein and are designed for C- or N-terminal fusion with genes of interest, or they encode an
organelle-targeted fluorescent protein

Plasmid Description Localization Colour References

pEH3 hph::Pgpd-egfp-TtrpC, C-terminal Cytoplasm Green Nowrousian et al. (2007a)
pYHN3 hph::Pgpd-eyfp-TtrpC, C-terminal Cytoplasm Yellow Rech et al. (2007)
pCHN3 hph::Pgpd-ecfp-TtrpC, C-terminal Cytoplasm Cyan Rech et al. (2007)
pRHN3 hph::Pgpd-DsRed-TtrpC, C-terminal Cytoplasm Red Engh et al. (2007b)
pMHN2 hph::Pgpd-mRFP1-TtrpC, C-terminal Cytoplasm Red Unpublished data
pTomato hph::Pgpd-tdTomato-TtrpC, C-terminal Cytoplasm Red Unpublished data
pmKalama1 hph::Pgpd-mKalama1-TtrpC, C-terminal Cytoplasm Blue Unpublished data
pN-EGFP hph::Pgpd-egfp-TtrpC, N-terminal Cytoplasm Green Unpublished data
pN-Tomato hph::Pgpd-tdTomato-TtrpC, N-terminal Cytoplasm Red Unpublished data
pYH2A hph::Pgpd-hh2a-eyfp-TtrpC Nucleus Yellow Rech et al. (2007)
pCH2B hph::Pgpd-hh2b-ecfp-TtrpC Nucleus Cyan Rech et al. (2007)
pDsRed-SKL nat::Pgpd-DsRed-SKL-TtrpC Peroxisome Red Elleuche and Pöggeler (2008)
pGFP-SKL phleo::Pgpd-egfp-SKL-TtrpC Peroxisome Green Ruprich-Robert et al. (2002)
pCW15 his3::Pccg1-sgfp-hex-1 Woronin body Green Engh et al. (2007b)
pEGFP-KDEL nat::Pgpd-Sppg1-egfp-KDEL-TtrpC ER Green Nowrousian et al. (2007a)
pDsRed-KDEL nat::Pgpd-pro41-DsRed-KDEL-TtrpC ER Red Nowrousian et al. (2007a)
 Sordaria macrospora, a Model System for Fungal Development
and ER appear to have a wild-type morphology,
although the proteins defective in these mutants
localize to these organelles (Engh et al. 2007b;
Nowrousian et al. 2007a).
A particular application of fluorescence
microscopy was developed for the quantification
of hyphal fusion events between different
S. macrospora strains (Rech et al. 2007). During
hyphal fusion, cytoplasmic mixing occurs, which
also enables mixing of genetic information by
exchange of nuclei between hyphal compart-
ments. Different strains were labelled with his-
tone fusion proteins H2A-YFP and H2B-CFP.
Following successful fusion, differently labelled
nuclei are present in one hyphal compartment,
and differently labelled histones are targeted to
29
Fig. 2.6. DIC and fluorescence
microscopic images of S. macro-
spora. Strains were transformed
with plasmids pCH2B (A), pGFP-
SKL (B), pYH2A (C), pTomato
(D), pCW15 (E) and pEGFP-
KDEL (F, G). H Ascus from a
sexual cross of different strains
transformed with pYH2A and
pCH2B, respectively. Note the
Mendelian segregation of the
fluorescent markers. Bar size is
the same for A–E and F–G,
respectively
the same nucleus. Subsequently, fluorescence mi-
croscopy shows both cyan and yellow fluores-
cence in one nucleus as a result of hyphal
fusion. Besides immunofluorescence and fluores-
cent proteins, vital fluorescent dyes have been
used for S. macrospora.
An example is Calcofluor white, an exceptional dye for
staining chitin in fungal cell walls. As such, it can be
applied for studying morphological changes, and its use
uncovered the hyperbranching phenotype of the mcm1
mutant (Nolting and Pöggeler 2006b). The DNA-staining
dye 40 ,6-diamidino-2-phenylindol (DAPI) was used for
studying meiotic and post-meiotic events (Storlazzi et al.
2003, 2008) as well as ascus development, which is closely
coupled to meiosis. For example, DAPI staining showed
abnormal ascus development in several S. macrospora
30 Ulrich Kück et al.
mutants like Dste12 and the pheromone receptor/phero-
mone precursor double mutant Dpre2/Dppg2 (Mayrhofer
et al. 2006; Nolting and Pöggeler 2006b). DAPI staining was
also used to show that the mouse striatin gene is able
to complement the developmental defects of the sterile
pro11 mutant (Pöggeler and Kück 2004).
In summary, a great variety of tools for fluores-
cence microscopy has been developed
for S. macrospora that allow not only protein local-
ization but also specialized experiments for further
studying the cell biology of this model organism.
D. Functional Genomics
In recent years, high-throughput methods for the
analysis of gene expression have been established
for a number of filamentous fungi. Most prominent
among these new techniques are microarrays that
allow the parallel analysis of gene expression for
(nearly) all genes within a genome (Nowrousian
et al. 2004a). Microarrays can be established from
cDNA sequences or, if the genome of an organism
has been sequenced, oligonucleotides as array
probes can be derived from genomic sequences;
and both types of arrays have been used for fila-
mentous fungi (Breakspear and Momany 2007). In
most cases, expression analyses with microarrays
are performed with the organisms for which the
array was developed (Nowrousian 2007). However,
if two species are closely related, it is possible to do
cross-species microarray hybridizations where the
targets that are used for hybridization are derived
from another organism than the one for which the
array was originally developed. This saves the effort
of setting up microarrays, especially for organisms
where the genome has not yet been sequenced.
S. macrospora was the first filamentous fungus for
which cross-species array hybridizations were
established (Nowrousian et al. 2005). Cross-species
hybridizations were performed first using cDNA
microarrays for N. crassa, a close relative of
S. macrospora with 90% nucleic acid sequence
identity within coding regions (Nowrousian et al.
2004b). It was subsequently shown that these cross-
species hybridizations could also be done with
oligonucleotide microarrays from N. crassa that
carry 70mer oligonucleotides as probes (Nowrou-
sian et al. 2007a).
Gene expression analysis with microarrays can
serve several purposes, for example: (1) the identi-
fication of differentially expressed genes for further
studies, and (2) the analysis of genetic networks by
making use of expression data as a molecular phe-
notype (Nowrousian 2007). Both aspects were stud-
ied in S. macrospora by comparing gene expression
in developmental mutants and a wild-type strain
(Nowrousian et al. 2005; Pöggeler et al. 2006b;
Nowrousian et al. 2007a). Microarray analysis was
performed using mutants pro1, pro11, pro22,
pro41, and DSmta-1, all of which produce only
protoperithecia (see Sects. IV, VI); and a number
of genes were identified that are up- or down-regu-
lated in the mutants compared to the wild type.
Among these are the genes for the pheromone
precursors ppg1 and ppg2, the putative lectin-
encoding gene tap1, a gene that was subsequently
shown to encode the fruiting body-specific protein
APP and the melanin biosynthesis genes pks and
sdh. These genes were further studied to decipher
the details of their expression patterns and their
roles in fruiting body development (Nowrousian
and Cebula 2005; Mayrhofer et al. 2006; Engh
et al. 2007a; Nowrousian et al. 2007b). Additionally,
overall expression patterns in the mutants were
used as molecular phenotypes to establish a genetic
network of pro genes (see also Sect. VI.E): expres-
sion patterns in mutants pro1, pro11 and pro22 are
more similar to each other than to the DSmta-1
mutant, indicating that Smta-1 may act in parallel
to these pro genes (Nowrousian et al. 2005;
Pöggeler et al. 2006b). Furthermore, it was shown
that pro41 acts genetically downstream of the pro1
gene (Nowrousian et al. 2007a).
Taken together, S. macrospora was among the
first filamentous fungi for which microarray
experiments were performed; and, in recent
years, the results of these high throughput
analyses have identified developmentally regu-
lated genes and aided analysis of genetic networks
that control fruiting body formation.
VI. Developmental Biology and Components
of Signalling Pathways
There is increasing evidence that both external and
internal signals are essential for fungal fruiting
body development. In several filamentous ascomy-
cetes these signals have been shown to be trans-
duced by conserved signal transduction pathways.
The components involved can be subdivided into
receptors that receive the signal, components that
 Sordaria macrospora, a Model System for Fungal Development
transmit the signal into the cell, and nuclear tran-
scription factors that regulate gene expression
(Pöggeler et al. 2006a). The molecular mechanisms
underlying sexual developmental processes in S.
macrospora are only poorly understood. However,
some of the key components of the signalling cas-
cades from the cell surface to the nucleus have been
genetically characterized in S. macrospora. More-
over, novel developmental proteins were discov-
ered that might function further downstream of
conserved signalling pathways, ensuring cell-spe-
cific differentiation.
A. Pheromones and Pheromone Receptors
Male and female fertility of heterothallic filamen-
tous ascomycetes depends on interactions of pher-
omones with their specific receptors. Pheromone
precursor genes encoding two different types of
pheromones have been isolated from heterothallic
filamentous ascomycetes. One of the precursor
genes encodes a polypeptide containing multiple
repeats of a putative pheromone sequence bor-
dered by protease processing sites and resembles
the a-factor precursor gene of Saccharomyces cere-
visiae. The other gene encodes a short polypeptide
similar to the S. cerevisiae a-factor precursor.
The short precursor has a C-terminal CaaX motif
(C = cysteine, a = aliphatic, X = any amino acid
residue) expected to produce a lipopeptide phero-
mone with a C-terminal carboxy-methyl-isopreny-
lated cysteine. The two types of pheromone
precursor genes are present in the same genome
and expression of pheromone genes seems to occur
in a mating type-specific manner (Bobrowicz et al.
2002; Coppin et al. 2005).
When pheromone genes were deleted in het-
erothallic ascomycetes, male spermatia were no
longer able to fertilize the female partner, proving
that pheromones are crucial for the fertility of
male spermatia (Turina et al. 2003; Coppin et al.
2005; Kim and Borkovich 2006). Sensing of the
pheromone signal depends on (G) protein-cou-
pled seven transmembrane-spanning receptors
(GPCRs). Two genes, designated pre-1 and pre-2,
encode pheromone receptors similar to S. cerevi-
siae a-factor receptor (Ste3p) and a-factor recep-
tor (Ste2p), respectively, and have been shown to
be essential for mating type-specific directional
growth and fusion of trichogynes and female fer-
tility in Neurospora crassa (Kim and Borkovich
31
2004, 2006). Thus, in heterothallic filamentous
ascomycetes the function of pheromones and
their cognate receptor seems to be restricted to
the fertilization process.
Interestingly, genes encoding two different
pheromone precursors and two pheromone recep-
tors have also been identified in the homothallic
Sordaria macrospora (Pöggeler 2000; Pöggeler and
Kück 2001). The pheromone precursor gene ppg1 is
predicted to encode a a-factor-like peptide phero-
mone and the ppg2 gene an a-factor-like lipopep-
tide pheromone (Pöggeler 2000). The deduced gene
products of the S. macrospora pre1 and pre2 genes
are predicted to be seven transmembrane domain
proteins (Pöggeler and Kück 2001). For S. macro-
spora, it has been demonstrated that disruption of
the pheromone precursor ppg1 gene prevents the
production of the peptide pheromone, but does not
affect vegetative growth, fruiting-body, or asco-
spore development (Mayrhofer and Pöggeler
2005). Similarly, no effect on vegetative growth,
fruiting-body, and ascospore development was ob-
served in the single pheromone-mutant Dppg2 and
single receptor-mutants Dpre1 and Dpre2. Howev-
er, double-knockout strains lacking any compatible
pheromone/receptor pair (Dpre2/Dppg2, Dpre1/D
ppg1) and the double-pheromone mutant strain
(Dppg1/Dppg2) have a drastically reduced number
of perithecia and sexual spores, whereas deletion of
both receptor genes (Dpre1/Dpre2) almost
completely eliminates fruiting body and ascospore
formation (Mayrhofer et al. 2006). Taken together,
these results suggest that pheromones and phero-
mone receptors are involved in post-fertilization
events and are required for optimal sexual repro-
duction of the homothallic S. macrospora. More-
over, by heterologously expressing the S.
macrospora pre2 in Saccharomycs cerevisiae
MATa cells, lacking the Ste2p receptor, PRE2 was
shown to facilitate all aspects of the S. cerevisiae
pheromone pathway when activated by the Sor-
daria macrospora peptide pheromone (Mayrhofer
and Pöggeler 2005). Therefore, one may conclude
that the receptors encoded by pre2 and pre1 also
function as G protein-coupled receptors (GPCRs)
in S. macrospora.
B. Heterotrimeric G Proteins
Upon activation by GPRC receptors, heterotri-
meric G proteins catalyse the exchange of GDP
32 Ulrich Kück et al.
for GTP on the G protein a subunit. This leads to
dissociation of the bg subunits. Either Ga, or Gbg,
or both are then free to activate downstream effec-
tors. Signalling persists until GTP is hydrolysed to
GDP, and the subunits re-associate (Dohlman
2002). The genome of S. macrospora contains
three genes (gsa1, gsa2 and gsa3) encoding Ga
subunits (Kamerewerd et al. 2008). In addition
genes encoding the b-subunit and the g-subunits
of the G protein are present in S. macrospora
(Mayrhofer and Pöggeler 2005).
To explore the functional role of G protein a
subunits (GSA) in the sexual life cycle of this
fungus, knockout strains for all three gsa-genes
(Dgsa1, Dgsa2, Dgsa3) and double mutants were
generated. Phenotypic analysis of single and dou-
ble mutants showed that the genes for Ga subunits
contribute differently to sexual development.
While the Dgsa2 mutant revealed wild-type-like
fertility, Dgsa3 developed fruiting bodies, but the
ascospores had a drastically reduced germination
rate. Dgsa1 showed a delay in sexual development
and a 50% reduction in the number of fruiting
bodies. A more impaired phenotype can be
PPG2
PRE1
GSA2
Fig. 2.7. Pheromone signalling
pathway showing the function
of pheromones, pheromone
receptors and G protein alpha
subunits, as well as downstream
signalling components in the de-
velopment of perithecia (modi-
fied from Kamerewerd et al.
2008)
observed in Dgsa1Dgsa2 and Dgsa1Dgsa3 double
mutants. These mutants produce only protoper-
ithecia, indicating that all Ga subunits contribute
significantly to sexual development. To test
whether the pheromone receptors PRE1 and
PRE2 mediate signalling via distinct Ga subunits,
Dpre strains were crossed with all Dgsa strains.
The data obtained with double mutants carrying a
disrupted gsa gene together with a disrupted re-
ceptor gene indicated that the pheromone recep-
tors interact differently with GSA1 or GSA2 and
that GSA1 is the main component of a signal
transduction cascade downstream of the phero-
mone receptors. As shown in Fig. 2.7, the third Ga
protein, GSA3, seems to be involved in a parallel
signalling pathway but also contributes to fruiting
body development and fertility (Kamerewerd et al.
2008).
C. Adenylyl Cyclase
Activated Ga or Gbg subunits can regulate down-
stream effectors such as adenylyl cyclase and
PPG1 ?
PRE2 ?
GSA1 GSA3
? SAC1
STE12 ?
fruiting body ascospore
development germination
 Sordaria macrospora, a Model System for Fungal Development
mitogen-activated protein kinase (MAPK) cas-
cades. The fungal adenylyl cyclase is a soluble
enzyme that produces the second messenger
cyclic AMP (cAMP) from ATP. The cAMP signal-
ling pathway of fungi is involved in several impor-
tant processes including nutrient sensing, stress
response, metabolism, pathogenicity, and sexual
development (Lengeler et al. 2000; D’Souza and
Heitman 2001). Several lines of evidence indicate
that many of the known fungal G proteins influ-
ence the intracellular level of cAMP by either
stimulating or inhibiting adenylyl cyclase (Bölker
1998). In order to investigate a component that
acts supposedly downstream of the GSAs, the
S. macrospora gene encoding adenylyl cyclase
(sac1) was isolated. Deletion of sac1 prevents
cAMP synthesis. Moreover, a Dsac1 mutant exhi-
bits reduced fertility with a significant number of
the fruiting bodies that are embedded in the solid
media. A similar phenotype was observed in a
Dgna3 mutant of N. crassa. In addition, ascos-
pores derived from the Dsac1 mutant have a high-
ly reduced germination rate and this phenotype
also resembles that of Ga subunit 3 disruption
mutants from N. crassa (Kays et al. 2000) and S.
macrospora (Kamerewerd et al. 2008). Analysis of
the three Dgsa/Dsac1 double mutants indicates
that SAC1 acts downstream of GSA3, parallel to a
GSA1-GSA2 mediated signalling pathway (Kamer-
ewerd et al. 2008).
D. Transcription Factors
Signal transduction initiates morphogenesis by
finally activating transcription factors which in
turn activate or repress cell- or tissue-specific
expression of developmental genes. To elucidate
whether conserved fungal transcription factors
are involved in sexual development of S. macro-
spora, mcm1 and ste12 were functionally analysed.
The mcm1 gene encodes a putative homologue of
the Saccharomyces cerevisiae MADS box protein
Mcm1p and ste12 encodes a homeodomain similar
to the S. cerevisiae Ste12p, respectively (Nolting
and Pöggeler 2006a, b). Deletion of the mcm1
gene led to a pleiotropic phenotype, including re-
duced biomass, increased hyphal branching, and
reduced hyphal compartment length during vege-
tative growth, as well as sexual sterility. The mcm1
mutant was only capable of producing protoper-
ithecia, but was unable to form either ascospores or
33
perithecia and, thus, phenotypically resembled
pro-mutants of Sordaria macrospora (see Sect.
IV). A two-hybrid analysis demonstrated that
MCM1 may physically interact with itself and
with the a-domain mating-type protein MAT1-1-1
(Nolting and Pöggeler 2006b). The pleiotropic phe-
notype of S. macrospora Dmcm1 suggests that the
S. macrospora DMCM1 protein might be involved
in a wide range of functions via interaction with
diverse transcriptional regulators.
From a yeast two-hybrid screen, among sev-
eral other proteins, a putative homologue of the
Saccharomyces cerevisiae homeodomain protein
Ste12p was identified as an additional MCM1 in-
teraction partner (Nolting and Pöggeler 2006a). In
S. cerevisiae, Ste12p is known to be a transcription
factor acting downstream of Fus3p/Kss1 MAP
kinases. In haploid yeast cells, Ste12p is required
for the response to the mating pheromone pro-
duced by the opposite mating type and for inva-
sive growth in response to limited nutrients. In
diploid yeast cells, Ste12p regulates pseudohyphal
development in response to nitrogen starvation
(Madhani and Fink 1997; Gustin et al. 1998;
Roberts et al. 2000). Transcriptional regulation
of different classes of genes is thereby triggered
through interactions with different transcriptional
regulators. Ste12p forms a homodimer and binds
either Mcm1p or Mata1p to regulate pheromone-
responsive genes and cell-type-specific genes,
respectively, and with Tec1p to activate genes
required for filamentous growth (Dolan et al.
1989; Yuan et al. 1993; Bruhn and Sprague 1994;
Madhani and Fink 1997).
In Sordaria macrospora, two-hybrid and bio-
chemical studies showed that STE12 in addition to
MCM1 is also able to interact with the mating-type
protein MAT1-1-1. Analysis of a S. macrospora
Dste12 knockout mutant demonstrated that
STE12 is not needed for fruiting body formation
and vegetative growth but is involved in ascospor-
ogenesis. The S. macrospora Dste12 mutant is able
to form protoperithecia and perithecia, but the
latter contain a drastically reduced number of
asci with predominantly non-viable ascospores.
Particularly, cell walls of asci and ascospores
appear to be fragile (Nolting and Pöggeler
2006a). This implies that STE12, in addition to
its role in pheromone signal transduction, might
be involved in cell wall integrity of asci and ascos-
pores. To study the functional connections
between the GSA subunits and the STE12
34 Ulrich Kück et al.
transcription factor in S. macrospora, the pheno-
types of three DgsaDste12 double mutants were
analysed (Kamerewerd et al. 2008). While
Dgsa2Dste12 double mutants show a wild-type-
like formation of perithecia containing fragile
asci and ascospores, the Dgsa1Dste12 mutant
develops only a few fruiting bodies. Furthermore,
perithecia of the Dgsa1Dste12 mutant contain
only a few asci compared to the Dste12 single or
the Dgsa2Dste12 double mutant. The Dgsa3Dste12
mutant exhibits the most severe phenotype.
Protoperithecia are produced, but no mature
perithecia can be detected. The sterility of the
Dgsa3Dste12 mutant is a strong evidence for the
STE12 transcription factor being one of the key
regulators downstream of the pheromone
receptors.
E. Novel Developmental Proteins
As shown in Fig. 2.4 (see Sect. IV), more than 100
S. macrospora mutants have been described with
blocks at various stages of the developmental
cycle. For seven of these mutants, the affected
genes have already been identified: The wild-type
phenotype was restored in the mutants by trans-
formation with an indexed cosmid library (Pög-
geler et al. 1997a). Thus it was possible to isolate
the wild-type alleles of the mutated genes. Using
this experimental approach, novel developmental
genes were identified, most of which had not been
implicated in fungal development before, thus
validating a forward genetic approach.
The mutants that were complemented are
pro1, pro4, pro11, pro22, pro40, pro41, and per5,
all of which have a block at the stage of protoper-
ithecium formation (pro mutants) or produce
perithecia but no mature ascospores (per5). The
corresponding genes can be grouped according to
the functions of their gene products (Table 2.2).
In mutants pro4 and per5, the leu1 and acl1 genes
encoding b-isopropylmalate dehydrogenase and
ATP citrate lyase, respectively, are defective
(Nowrousian et al. 1999; Kück 2005). They are
involved in amino acid and fatty acid biosynthe-
sis, respectively, and their requirement for fruit-
ing body formation, but not for vegetative growth
may indicate an increased energy demand during
sexual development. Expression analyses showed
that both genes are under transcriptional control
during the developmental cycle of S. macrospora,
indicating a tight integration of metabolic activity
and developmental processes.
The first pro gene to be identified was pro1, a
gene encoding a zinc finger transcription factor
(Masloff et al. 1999, 2002). The gene product
belongs to the fungal-specific class of zinc cluster
proteins, the best known member of which is the
yeast transcriptional regulator Gal4 (MacPherson
et al. 2006). pro1 was the first gene encoding a
transcription factor of this class that was shown to
be essential for sexual development in filamentous
fungi. Since then, pro1 orthologues from several
other filamentous ascomycetes have been identified
that also have a role in development; however, there
seems to be a great diversity in the function of pro1
orthologues in that their roles can include functions
in vegetative sporulation or growth (Colot et al.
2006; Vienken and Fischer 2006).
The last four pro genes that were cloned en-
code proteins that are either membrane or mem-
brane-associated proteins and/or localize to
organelles. Among these is PRO11, a WD40 repeat
protein that associates with membranes (Pöggeler
and Kück 2004). It is a functional homologue of
the mammalian protein striatin as was demon-
strated by the ability of the mouse orthologue to
restore fertility in the pro11 mutant. This indi-
cates that PRO11 is a member of a protein family
with functionally conserved members from lower
to higher eukaryotes. Another putative membrane
or membrane-associated protein that is encoded
by a pro gene is PRO22, an orthologue of the
N. crassa protein HAM-2. The latter was shown
to be necessary for hyphal fusion in N. crassa and,
like the corresponding N. crassa mutant, the pro22
mutant is defective in hyphal fusion (Xiang et al.
2002; Rech et al. 2007).
Two identified organellar proteins are PRO40
and PRO41, that localize to the Woronin body and
the ER, respectively (Engh et al. 2007b; Nowrou-
sian et al. 2007a). PRO40 is a WW domain protein
and the first of its class that was shown to be
essential for fruiting body development. Addition-
ally, it is the first Woronin body protein for which
a role in development could be demonstrated. The
Woronin body is an organelle that is specific to
filamentous ascomycetes where it is necessary for
septal plugging to prevent loss of cytoplasm after
hyphal injury (Markham and Collinge 1987).
Woronin bodies have not been previously impli-
cated in fruiting body formation; however, detec-
tion of PRO40 (and its corresponding N. crassa
 Sordaria macrospora, a Model System for Fungal Development 35

Table 2.2. Developmental genes from S. macrospora. Localizations given in brackets are derived from sequence
homologies, the others were verified experimentally. The genes acl1 and leu1 complement mutants per5 and pro4,
respectively, all other genes carry the same name as the corresponding mutant

Gene Gene product/conserved domains Localization References

Primary metabolism
acl1 Subunit of the ATP citrate lyase Cytoplasm Nowrousian et al. (1999)
leu1 b-Isopropylmalate dehydrogenase (Cytoplasm) Kück (2005)
Secondary metabolism
fbm1 Dehydrogenase (polyketide biosynthesis) (Extracellular) Nowrousian (2009)
pks Polyketide synthase (melanin biosynthesis) (Cytoplasm) Engh et al. (2007b)
sdh scytalon dehydratase (melanin biosynthesis) (Cytoplasm) Engh et al. (2007b)
Transcription factors
mcm1 MADS-box transcription factor Nucleus Nolting and Pöggeler (2006b)
pro1 C6 zinc finger transcription factor (Nucleus) Masloff et al. (1999)
Smta-1 HMG domain transcription factor (Nucleus) Pöggeler et al. (1997b)
ste12 Homeodomain/zinc finger transcription factor (Nucleus) Nolting and Pöggeler (2006a)
Pheromones and pheromone receptors
ppg1 Peptide pheromone Extracellular Mayrhofer and Pöggeler (2005)
ppg2 Lipopeptide pheromone (Extracellular) Mayrhofer et al. (2006)
pre1 Pheromone receptor (Plasma membrane) Mayrhofer et al. (2006)
pre2 Pheromone receptor (Plasma membrane) Mayrhofer et al. (2006)
Signal transduction
gsa1 G protein a-subunit (Membrane-associated) Kamerewerd et al. (2008)
gsa2 G protein a-subunit (Cytoplasm) Kamerewerd et al. (2008)
gsa3 G protein a-subunit (Membrane-associated) Kamerewerd et al. (2008)
sac1 Adenylate cyclase (Cytoplasm) Kamerewerd et al. (2008)
Membrane proteins and membrane-associated proteins
pro11 WD40-repeat protein Membrane-associated Pöggeler and Kück (2004)
pro22 Membrane protein (Membrane) Rech et al. (2007)
Organellar proteins
pro40 WW domain protein Woronin body Engh et al. (2007a)
pro41 Membrane protein ER membrane Nowrousian et al. (2007)

orthologue SO that also localizes to septal plugs
and is essential for sexual development) makes it
tempting to speculate about as yet unsuspected
roles for this fungal-specific organelle (Engh
et al. 2007b; Fleißner and Glass 2007).
The last pro gene to be isolated was pro41 that
encodes a small protein of the ER membrane
(Nowrousian et al. 2007a). PRO41 orthologues
exist only in filamentous ascomycetes and the
S. macrospora gene was the first of these to be
functionally characterized. Similar to the other
pro genes, pro41 is dispensable for overall vegeta-
tive growth but is necessary for the formation of
mature fruiting bodies. Expression studies showed
that it is transcriptionally upregulated during fruit-
ing body formation; and microarray analyses of
single and double mutants indicated that it acts
genetically downstream of the transcription factor
gene pro1. The identification of the development-
specific proteins PRO40 and PRO41 throws a spotlight
on the role of organelles in fungal development.
Future studies should include the identification
of interaction partners of the proteins as well as
further analysis of the genetic interactions between
the genes to unravel metabolic and signalling net-
works that integrate subcellular functions with
multicellular development.
Another interesting aspect that came to light
during the analysis of the pro-mutants is the fact
that there might be a connection between hyphal
fusion events and the ability to form sexual
structures: Mutants pro22 and pro40 are greatly
impaired in their ability to form anastomoses
between vegetative hyphae, and the same is true
for the corresponding N. crassa mutants ham-
2 and soft, both of which were initially identified
in screens for hyphal fusion mutants (Xiang et al.
2002; Fleißner and Glass 2007; Rech et al. 2007).
There are a number of hyphal fusion events
necessary for sexual development. These include
36 Ulrich Kück et al.
fusion of sub-apical cells in crozier formation as
well as (putative) fusion of hyphae that form the
outer perithecial shell. In heterothallic ascomy-
cetes like N. crassa, an additional fusion event
has to occur to allow fertilization; however, the
SO protein is not necessary for this specific type of
fusion (Fleissner et al. 2005). Thus, the connection
between hyphal fusion events and fruiting body
development has yet to be discovered and poses
one of the most challenging future questions in
fungal biology.
VII Conclusions
Sordaria macrospora has a long-standing history as
a classic genetic model system for conventional
tetrad analysis. The application of several molecular
tools, such as DNA mediated transformation, site-
specific recombination or functional genomics to
this filamentous fungus makes it an ideal experi-
mental system to uncover the details of genetic
interactions controlling developmental plasticity.
The rapid and inexpensive genetic analysis of
developmental mutants with distinct and defined
morphological defects will increase our knowledge
of multicellular differentiation processes in eukar-
yotes.
Acknowledgements. We thank Ms. Gabriele Frenßen-
Schenkel for artwork, Ms. Melanie Mees for typing the
manuscript and Dr. Sandra Masloff for preparing scanning
electron microscopic images from developmental
mutants. The work of the authors is substantially sup-
ported by funding from the Deutsche Forschungsge-
meinschaft (Bonn-Bad Godesberg, Germany).
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Zickler D (1977) Development of the synaptonemal com-
plex and the ‘‘recombination nodules’’ during meiotic
prophase in the seven bivalents of the fungus
Sordaria macrospora Auersw. Chromosoma 61:
289–316
3 Inteins – Selfish Elements in Fungal Genomes

SKANDER ELLEUCHE1, STEFANIE PÖGGELER1

CONTENTS excised at the protein level (Fig. 3.1). By analogy

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 to introns and exons, the terms intein and extein
A. General Characteristics of Inteins . . . . . . . . . . 42 have been proposed, for internal protein sequence
B. Protein Splicing Mechanism . . . . . . . . . . . . . . . . 44 and external protein sequence of the precursor,
II. Inteins in Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 respectively. Upstream exteins are termed the
A. VMA1 Inteins of Saccharomycete Yeasts . . . . 46
B. PRP8 Inteins in Fungi . . . . . . . . . . . . . . . . . . . . . . . 46 N-extein and downstream exteins the C-extein.
1. Distribution of PRP8 Inteins The post-translational process that excises the
in Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 internal region from a precursor protein with
2. Activity of Fungal PRP8 Inteins . . . . . . . . 49 subsequent ligation of the N- and C-extein is
C. Other Fungal Inteins . . . . . . . . . . . . . . . . . . . . . . . . . 50 termed protein splicing (Perler et al. 1994). Thus,
III. Mobility, Evolution, and Domestication
of Inteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 protein splicing is a processing event in which the
A. Mobility of Fungal Inteins . . . . . . . . . . . . . . . . . . . 52 expression of a single gene results in production
B. Evolution of Fungal Inteins . . . . . . . . . . . . . . . . . 53 of two stable proteins, the mature protein and the
C. Domestication of Fungal Inteins . . . . . . . . . . . . 54 intein (Fig. 3.1).
IV. Application of Inteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
A. Inteins and Their Application in
Protein–Protein Interaction Studies . . . . . . . . 54 Protein splicing elements were first described in fungi. In
B. Regulation of Protein-Splicing Activity . . . . 55 1990, two groups reported an in-frame insertion in the
C. Intein-Mediated Protein Purification . . . . . . . 56 Saccharomyces cerevisiae VMA1 gene encoding a vacuolar
D. Screening Systems for Protein-Splicing membrane H+-ATPase (Hirata et al. 1990; Kane et al.
Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 1990). The nucleotide sequence of the S. cerevisiae VMA1
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 gene predicts a polypeptide of 1071 amino acids (aa) with a
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 calculated molecular mass of 118 kDa, which was much
larger than the 67 kDa value estimated from sodium dode-
cyl sulfate–polyacrylamide (SDS-PAGE) gels. The N- and
C-terminal regions of the deduced sequence were shown to
be very similar to those of the catalytic subunits of vacuo-
lar membrane H+-ATPases of other organisms, while the
I. Introduction internal region containing 454 amino acid residues dis-
played no detectable sequence similarities to any known
ATPase subunits; instead it exhibited similarity to a yeast
Since the late 1970s it has become clear that the endonuclease encoded by the HO gene. Subsequently, it
coding sequence of many eukaryotic genes is dis- was demonstrated that the insertion was still present in the
rupted by genetic elements called intervening mRNA and translated together with the VMA1 protein,
sequences, which must be removed prior to host and that the insertion spliced itself out of the protein
gene function. Intervening sequences can be clas- (Kane et al. 1990).
sified into intron and intein. Introns are excised
from the primary RNA transcript by a process By now, more than 350 inteins have been recog-
termed splicing. In contrast to introns, inteins nized in the genomes of bacteria, archaea, and
are internal in-frame insertions transcribed and eukaryotes, as well as in viruses. Inteins have
translated together with their host protein and been identified predominantly in prokaryotes,
but are also found in greater than 70 eukaryotic
1
taxa. Eukaryotic inteins are encoded within the
Abteilung Genetik eukaryotischer Mikroorganismen,
Institut für Mikrobiologie und Genetik, Georg-August-Uni-
nuclear genomes of fungi, as well as within the
versität Göttingen, Grisebachstrasse 8, 37077 Göttingen, nuclear genomes and plastomes of some unicellu-
Germany; e-mail: spoegge@gwdg.de lar algae. For current information, see the intein

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
42 Skander Elleuche and Stefanie Pöggeler
N-Extein
coding sequence
DNA:
RNA: 5'
NH2
Protein: N-Extein
NH2
COOH
Protein:
Mature protein
Fig. 3.1. Protein splicing of inteins. The intein coding
sequence is transcribed into mRNA and translated to a
non-functional protein precursor, which then undergoes
registry InBase at http://www.neb.com/neb/
inteins.html (Perler 2002). According to accepted
nomenclature, intein names include a genus and
species designation (abbreviated to three letters)
and a host gene designation. For example the
S. cerevisiae VMA1 intein is called Sce VMA1.
When a protein contains more than one intein,
they are given Roman numerals (Perler et al. 1994).
This review focuses on fungal inteins and
includes the characteristics, as well as the distri-
bution of fungal inteins. It summarizes recent
progress in understanding the protein splicing
mechanism, how inteins evolve, and how they
can be used for technical applications.
A. General Characteristics of Inteins
Inteins are classified into two groups: large and
minimal (mini-) inteins, depending on whether or
not they contain a homing endonuclease domain
(Liu 2000; Fig. 3.2). Homing endonucleases are
Intein C-Extein
coding sequence coding sequence
Transcription
3'
Translation
Intein
Precursor protein
COOH C-Extein
Protein Splicing
+ NH2 COOH
Excised intein
a self-catalyzed rearrangement in which the intein is
excised and the exteins are joined to yield the mature
protein
encoded by open reading frames that are embedded
within introns and inteins (Chevalier and Stoddard
2001; Stoddard 2005). They are site-specific, double-
strand DNA endonucleases that promote the lateral
transfer of their own coding region and flanking
sequences between genomes by a recombination-
dependent process known as ‘homing’ (Fig. 3.3).
Genomic DNA encoding an intein-free allele of the
potentially intein-carrying gene is cut by the homing
endonuclease, and during DNA repair, the intein is
subsequently copied into the empty allele (Gimble
and Thorner 1993; Gimble 2000). The nomenclature
of homing endonucleases is patterned after that of
restriction enzymes. A three-letter, genus- and spe-
cies-specific designation is followed by a Roman
numeral to distinguish multiple enzymes originating
from a single organism. Intron-encoded endonu-
cleases are characterized by the prefix I (for intron),
whereas intein-derived endonucleases are character-
ized by the prefix PI (for protein insert; i.e. the
endonuclease of Sce VMA1 is termed PI SceI; Belfort
and Roberts 1997).
 Inteins – Selfish Elements in Fungal Genomes
Homing endonuclease
C D
Large intein:
Linker region
Mini-intein:
N-splicing domain
A N2 B N4
NH2 N-Extein
C TXXH
S Q S
A T
(numbering of amino acid residues)
-2 -1 12
Fig. 3.2. Conserved elements in a large intein and mini-
intein. The white areas A, N2, B, N4, C, D, E, F and G are
conserved intein motifs identified by Pietrokovski (Pie-
trokovski 1994, 1998b) and Perler et al. (1997). The
exteins are illustrated in black and the intein sequence
Four families of homing endonucleases containing highly
conserved amino acid sequences have been identified;
including the LAGLIDADG, the GIY-YIG, the His-Cys
box, and the H-N-H endonucleases (Chevalier and
Stoddard 2001). Fungal intein endonucleases belong to
the LAGLIDADG family and are characterized by two
copies of the conserved LAGLIDADG motif (Belfort and
Roberts 1997).
Mini-inteins are not bifunctional and contain only
a splicing domain without endonuclease function.
Because several splicing-efficient mini-inteins
have been engineered from large inteins by delet-
ing the central endonuclease domain, it is clear
that the endonuclease domain is not involved in
protein splicing (Chong and Xu 1997; Derbyshire
et al. 1997; Shingledecker et al. 1998). The splicing
domain of large inteins seems to be split by the
endonuclease domain into N-terminal and C-ter-
minal subdomains. Both intein subdomains con-
tain conserved blocks of amino acids: blocks A,
N2, B, and N4 for the N-terminal subdomains, and
blocks G and F for the C-terminal subdomains
(Pietrokovski 1994, 1998b; Perler et al. 1997;
Fig. 3.2). These domains can also be identified in
mini-inteins. However, motifs C, D, E, and H of
the endonuclease domain are missing; instead
they have a small linker region (Fig. 3.2). In addi-
tion, all known inteins have a Ser, Cys, or Ala at
the N terminus and end in His-Asn, or in His-Gln.
There is no conserved residue preceding the intein
43
E H
C-splicing domain
F G
C-Extein COOH
HN C
n-1 n +1 +2
in grey. The site of insertion of the homing endonuclease
and the linker region in large and mini-inteins, respec-
tively, is indicated by the dark vertical line. Conserved
amino acid residues of the intein and the C-extein are
indicated below
at the C-terminus of the N-extein, but all inteins
have an invariant Ser, Thr, or Cys at the N-
terminus of the C-extein (Perler 2002). According
to the most common amino acid numbering sys-
tem in intein precursors, minus and plus signs are
used to designate N-extein and C-extein residues,
respectively. Numbering begins at the N-terminus
of the intein and C-extein, and proceeds to the C-
terminus. Numbering of the N-extein begins at its
C-terminus and proceeds to its N-terminus (Saleh
and Perler 2006; Fig. 3.2).
Three-dimensional structures of inteins reveal
that the N- and C-terminal splicing domains form
a common horseshoe-like 12-b-strand scaffold
termed the Hedgehog/(Hint) module (Koonin
1995; Hall et al. 1997; Klabunde et al. 1998; Perler
1998; Ding et al. 2003; Sun et al. 2005).
Hedgehog proteins are essential signaling molecules for
animal embryonic development and possess an autopro-
cessing activity that results in an intramolecular cleavage
of the full-length Hedgehog protein and covalent attach-
ment of a cholesterol moiety to the newly generated
amino-terminal fragment. Cholesterol anchors the signal-
ing domain to the cell surface (Beachy et al. 1997; Ingham
2001). Until now, Hedgehog proteins have been described
only in metazoa.
Interestingly, the developmental protein GmGIN1
was identified in the arbuscular mycorrhizal fungus
Glomus mossae as having an N-terminal domain
44 Skander Elleuche and Stefanie Pöggeler
Transcription & Translation
intein-free
allele
intein-containing
allele
large intein
with homing endonuclease
Hydrolysis of recognition sequence
Double Strand Break Repair
Fig. 3.3. Intein homing. The large intein encodes an endo-
nuclease that in heterozygous cells recognizes a recognition
sequence in the intein-less allele on the homologous chro-
mosome. The recognition site is hydrolyzed by the intein-
encoded endonuclease, while the chromosome carrying
that shares sequence similarity with a novel family
of GTP-binding proteins, while the C-terminus has
a striking homology to the C-terminal part of the
Hedgehog protein family from metazoa. It is there-
fore hypothesized that the N-terminal part of
GmGIN1 might be activated through a similar
autoprocessing mechanism, as was demonstrated
for Hedgehog proteins (Requena et al. 2002). In
addition to Hedgehog proteins, fungal inteins also
share sequence and structure properties with
bacterial intein-like sequence (BIL) Amitai et al. 2003.
Combining all these characteristics, four main
criteria help to differentiate true inteins from other
in-frame insertions: (i) an intein-containing pro-
tein has a sequence lacking in homologs of other
organisms, (ii) the intervening sequence is over 100
aa, (iii) it contains the conserved intein motifs A, B,
F, and G, and (iv) the observed protein product is
the same size as the predicted open reading frame
without the intein (Perler et al. 1997).
recognition
sequence
homing
endonuclease
the intein is protected from being hydrolyzed. After cleav-
age, the recominational repair system of the cell is stimu-
lated and the intein-containing chromosome is used as a
template for repair. The result of intein homing is a homo-
zygote with two copies of the intein-containing gene
B. Protein Splicing Mechanism
The post-translational removal of the intein is an
autocatalytic process that depends only on the
information of the intein plus the first downstream
extein residue (Fig. 3.1). Protein splicing is ex-
tremely rapid, and to date, precursors have not
been identified in native systems. The first direct
evidence for protein splicing was demonstrated for
an intein derived from the thermostable DNA po-
lymerase of Pyrococcus species GB-D. The intein
from Pyrococcus was cloned into a foreign gene,
and the purified precursor was shown to be capa-
ble of in vitro protein splicing in the absence of
other proteins or cofactors (Xu et al. 1993).
Protein splicing typically involves four steps:
(i) an N-O or N-S acyl shift at the N-extein/intein
junction, (ii) transesterification, which transfers
the N-extein to the side-chain of the first residue
(+1) of the C-extein, (iii) cyclization of a
 Inteins – Selfish Elements in Fungal Genomes
conserved asparagine residue at the C-terminus of
the intein and cleavage of the peptide bond, result-
ing in the release of the intein, and (iv) rearrange-
ment to a peptide bond of the ester/thioester bond
linking the N- and C-exteins. The details of the
chemical process involved in protein splicing have
been comprehensively described and reviewed
(Liu 2000; Noren et al. 2000; Paulus 2000; Gogar-
ten et al. 2002; Saleh and Perler 2006; Staroka-
domskyy 2007).
Several inteins exist as two fragments and are
encoded by two separately transcribed and trans-
lated genes. These so-called split inteins self-asso-
ciate and catalyze protein-splicing activity in
trans. The first native split intein capable of pro-
tein trans-splicing was identified in the catalytic
subunit alpha of DNA polymerase III DnaE of the
cyanobacterium Synechocystis sp. strain PCC6803
(Wu et al. 1998b).
The N- and C-terminal halves are encoded by two separate
genes, dnaE-n and dnaE-c, respectively. These two genes are
located 745 226 bp apart in the genome and are on opposite
DNA strands (Wu et al. 1998b).
IN
A N2 B N4
NH2 N-Extein
C HN C
IN
A N2
NH2 N-Extein
C NH2
NH2 N-Extein C-Extein COOH
mature protein
Fig. 3.4. Protein splicing in trans. The precursor is
encoded by two genes: one gene encodes the N-extein
and the N-terminal intein (IN), the other gene encodes
the C-terminal part of the intein (IC) and the C-extein.
45
Currently, native split inteins have been identified
in diverse cyanobacteria and the archaeon
Nanoarchaeum equitans (Wu et al. 1998b; Caspi
et al. 2003; Liu and Yang 2003; Choi et al. 2006;
Dassa et al. 2007), but so far they have not been
found in fungi. Trans-splicing inteins have, how-
ever, been artificially engineered from cis-splicing
bacterial and fungal inteins (Mills et al. 1998;
Southworth et al. 1998; Mootz and Muir 2002;
Elleuche and Pöggeler 2007). In the majority of
cases, naturally or artificially split inteins are sepa-
rated between motifs B and F resulting in N-ter-
minal intein fragments (IN) of 25–40 aa and C-
terminal intein fragments (IC) of
100 aa
(Fig. 3.4). In addition, it was demonstrated that
inteins can be artificially split at other sites. The
Ssp DnaB mini-intein can be split at different loop
regions between b-strands and still maintain the
ability to splice in trans. Even a three-piece split
intein can function in protein trans-splicing (Sun
et al. 2004). Naturally split inteins, as well as engi-
neered split inteins can be used for various applica-
tions in protein chemistry, e.g. protein purification,
Transcription & Translation
IC
F G
COOH NH2 C-Extein COOH
Association
IC
F G
B N4
C-Extein COOH
HN C
COOH
Protein Splicing
F G
A N2 B N4
COOH
NH2
NH2
COOH
associated intein
Both genes are separately transcribed and translated, the
intein fragments associate and form a functional intein.
N- and C-exteins become ligated to the mature protein and
the intein fragments usually remain associated in solution
46 Skander Elleuche and Stefanie Pöggeler
regulation of protein activity, and analysis of
protein–protein interaction (see Sect. IV).
II. Inteins in Fungi
In fungal organisms, only cis-splicing inteins have
been detected, and all are encoded by nuclear
genes (Poulter et al. 2007). According to InBase
(Perler 2002), to date 73 inteins have been
detected in fungi. They have been identified in
chytrids, zygomycetes, ascomycetes, and basidio-
mycetes. Most of them are embedded in homologs
of the S. cerevisiae VMA1 gene or within the prp8
gene, but they can also be found in genes encoding
glutamate synthases, chitin synthases, threonyl-
tRNA synthetases, and subunits of DNA-directed
RNA polymerases (Poulter et al. 2007).
A. VMA1 Inteins of Saccharomycete Yeasts
The VMA1 gene of many saccharomycete yeasts
consists of a single open reading frame that com-
prises two independent regions of genetic informa-
tion for VMA1, the catalytic 67-kDa subunit of the
vacuolar H+-ATPase, and for an intein varying in
length from 394 to 517 residues (Table 3.1). Alleles
of the VMA1 gene lacking the intein element are
present in the fission yeast Schizosaccharomyces
pombe and in filamentous ascomycetes, including
Neurospora crassa (Bowman et al. 1988; Ghislain
and Bowman 1992). In addition several saccharo-
mycete yeast species do not contain a VMA1 intein.
Koufopanou et al. (2002) surveyed 24 species of
saccharomycete yeasts and detected VMA1 inteins
in 14 species that diverged in amino acid sequence.
All inteins found in VMA1 genes of saccharomycete
yeasts are large inteins and contain two separate
domains: a splicing domain and an endonuclease
domain (Table 3.1). The catalytic subunit of the
vacuolar proton pump is responsible for the ATP
hydrolysis that is coupled to the pumping of pro-
tons into a variety of intracellular acidic compart-
ments, including the fungal vacuole (Anraku et al.
1992). Under laboratory conditions, VMA1 mutants
of Saccharomyces cerevisiae are viable but lack the
vacuolar H+-ATPase and are defective in vacuolar
acidification. Moreover, they are calcium-sensitive
and exhibit low spore viability as well as slow
growth (Hirata et al. 1990). Thus, under natural
conditions the VMA1 gene seems to be essential
for yeast cell growth.
Until now, all identified VMA1 inteins are
inserted at an identical insertion site. The best-
characterized VMA1 intein is Sce VMA1 from
S. cerevisiae. As shown by crystal structure, the
protein splicing domain of Sce VMA1 is structurally
related to bacterial mini-inteins and to the Drosoph-
ila Hedgehog protein autoprocessing domain (Koo-
nin 1995; Duan et al. 1997; Hall et al. 1997;
Pietrokovski 1998b; Moure et al. 2002). Site-direct-
ed mutagenesis experiments and deletion of the
endonuclease domain show that endonuclease ac-
tivity is not required for protein splicing (Gimble
and Stephens 1995; Chong and Xu 1997). The two-
domain structure (self-splicing and endonuclease
domain) of PI-SceI was confirmed by X-ray crystal-
lography. However, despite the apparent structural
autonomy of the protein splicing and endonuclease
domains, the two domains appear to collaborate by
interacting with the homing site DNA (Duan et al.
1997; Moure et al. 2002; Werner et al. 2002). Fur-
thermore, it was shown that the two intein domains
are functionally independent and have separate
evolutionary origins (Dalgaard et al. 1997; Derby-
shire et al. 1997; Okuda et al. 2003; see Sect. III).
B. PRP8 Inteins in Fungi
After the discovery of the VMA1 intein, a second,
non-allelic intein in fungi was identified within the
PRP8 protein of the basidiomycete Cryptococcus
neoformans. Many fungal inteins have also been
found at an allelic insertion site in various fungi;
some of these have been characterized experimen-
tally in detail.
1. Distribution of PRP8 Inteins in Fungi
A nuclear-encoded intein has been identified
inside the prp8 gene in two strains (Cn 3511 and
JEC 21) of the human pathogen C. neoformans
(Butler et al. 2001). The PRP8 protein is a major
component of the spliceosome and is highly con-
served among eukaryotes (Grainger and Beggs
2005). Therefore, the PRP8 protein is essential
for cell viability (Luo et al. 1999) and PRP8 inteins
must be active, because inactivity leads to host
lethality. The Cne PRP8 intein (Table 3.2) is a
mini-intein composed of only 172 aa and
lacks the endonuclease domain, present in the
 Inteins – Selfish Elements in Fungal Genomes 47

Table 3.1. Characteristics of VMA1 inteins inserted in the vacuolar ATPase, subunit A

Species/intein Intein size, Activity References

endonuclease
Saccharomyces cerevisiae/Sce VMA1 454 aa, + Experimental Hirata et al. (1990), Kane et al. (1990)
S. cerevisiae DH1-1A/Sce-DH1-1A VMA1 454 aa, + Theoretical Gimble (2001)
S. cerevisiae OUT7091/Sce-OUT7091 VMA1 454 aa, + Theoretical Okuda et al. (2003)
S. cerevisiae OUT7112/Sce-OUT7112 VMA1 454 aa, + Theoretical Okuda et al. (2003)
S. castellii CBS4309/Sca-CBS4309 VMA1 517 aa, + Theoretical Koufopanou et al. (2002),
Okuda et al. (2003)
S. castellii IFO1992/Sca-IFO1992 VMA1 517 aa, + Theoretical Koufopanou et al. (2002),
Okuda et al. (2003)
S. cariocanus/Scar VMA1 454 aa, + Theoretical Perler (2002)
S. dairenensis/Sda VMA1 501 aa, + Theoretical Koufopanou et al. (2002),
Okuda et al. (2003)
S. exiguous/Sex VMA1 499 aa, + Theoretical Koufopanou et al. (2002),
Okuda et al. (2003)
S. pastorianus/Spa VMA1 454 aa, + Theoretical Okuda et al. (2003)
S. unisporus/Sun VMA1 414 aa, + Theoretical Koufopanou et al. (2002),
Okuda et al. (2003)
Kazachstania exiguus/Kex VMA1 502 aa, + Theoretical Koufopanou et al. (2002),
Okuda et al. (2003)
Candida tropicalis/Ctr VMA1 471 aa, + Experimental Gu et al. (1993), Steuer et al. (2004)
C. glabrata/Cgl VMA1 415 aa, + Theoretical Okuda et al. (2003)
Debaryomyces hansenii/Dha VMA1 394 aa, + Theoretical Dujon et al. (2004)
Pichia stipitis/Pst VMA1 449 aa, + Theoretical Bakhrat et al. (2006),
Jeffries et al. (2007)
Kluyveromyces lactis CBS683/Kla-CBS683 VMA1 410 aa, + Theoretical Koufopanou et al. (2002),
Okuda et al. (2003)
K. lactis IFO1267/Kla-IFO1267 VMA1 410 aa, + Theoretical Okuda et al. (2003)
K. lactis NRRLY1140/Kla-NRRLY1140 VMA1 410 aa, + Theoretical Dujon et al. (2004)
Vanderwaltozyma polyspora/Vpo VMA1 433 aa, + Theoretical Koufopanou et al. (2002)
Lodderomyces elongisporus/Lel VMA1 421 aa, + Theoretical Perler (2002)
Torulaspora globosa/Tgl VMA1 456 aa, + Theoretical Koufopanou et al. (2002)
T. pretoriensis/Tpr VMA1 455 aa, + Theoretical Koufopanou et al. (2002)
Zygosaccharomyces bailii/Zba VMA1 456 aa, + Theoretical Koufopanou et al. (2002)
Z. bisporus/Zbi VMA1 450 aa, + Theoretical Koufopanou et al. (2002)
Z. rouxii/Zro 450 aa, + Theoretical Koufopanou et al. (2002)
Schizosaccharomyces japonicus/Sja VMA1 476 aa, + Theoretical Perler (2002)

experimentally characterized VMA1 intein of
S. cerevisiae (Butler et al. 2001; see Sect. II.A).
Isolates of C. neoformans have been classified
into five different serotypes and three different
varieties. They are named C. neoformans var. gru-
bii (serotype A), var. gattii (serotypes B, C), var.
neoformans (serotype D), and a hybrid of sero-
types A and D (serotype AD; Nelson and Lodes
2006; Ito-Kuwa et al. 2007).
Varieties grubii and neoformans are distributed all over the
world, while serotypes B and C are essentially restricted to
tropical and subtropical regions (Granados and Castaneda
2005). Due to the evolutionary distance of C. neoformans
var. gattii, Kwon-Chung et al. (2002) proposed separating
the fungus from C. neoformans and naming it C. gattii.
Liu and Yang (2004) detected highly conserved
PRP8 inteins with a size range of 170–172 aa in
the genomes of different C. neoformans serotypes.
In addition to sequence variations within the
intein, PRP8 inteins from different serotypes
also differ in their sizes. While PRP8 inteins of
serotype A are 172 aa in length, PRP8 inteins of
serotype B/C are 170 aa and inteins of serotype D
are 171 aa in length (Butler and Poulter 2005).
C. neoformans serotype AD carries two distinct
PRP8 inteins, indicating that strains of this sero-
type may be diploid (Poulter et al. 2007).
Besides the PRP8 mini-inteins identified in
C. neoformans and C. gattii, a large intein was
found within the PRP8 protein of Cryptococcus
laurentii (Butler and Poulter 2005). To date, no
48 Skander Elleuche and Stefanie Pöggeler

Table 3.2. Characteristics of PRP8 inteins inserted in a subunit of the spliceosome

Species/intein Intein size, Activity References

endonuclease
Aspergillus brevipes FRR2439/Abr PRP8 165,  Theoretical Butler et al. (2006)
A. fumigatus FRR0163/Afu-FRR0163 PRP8 820, + Experimental Liu and Yang (2004)
A. fumigatus var. ellipticus Af293/Afu- 819, + Theoretical Poulter et al. (2007)
Af293 PRP8
A. giganteus NRRL6136/Agi PRP8 167,  Theoretical Butler et al. (2006)
A. nidulans FGSC A4/Ani-FGSCA4-PRP8 605, + Experimental Liu and Yang (2004)
A. viridinutans FRR0577/Avi PRP8 169,  Theoretical Butler et al. (2006)
Batrachochytrium dendrobatidis JEL423/Bde- 465, + Theoretical Perler (2002)
JEL423 PRP8-1
B. dendrobatidis JEL423/Bde-JEL423 PRP8-2 465, + Theoretical Perler (2002)
Botrytis cinerea B05.10/Bci PRP8 838, + Theoretical Butler et al. (2006)
Cryptococcus gattii (serotype C/B)/Cga PRP8 170,  Theoretical Liu and Yang (2004),
Butler and Poulter (2005)
C. laurentii CBS139/Cla PRP8 522, + Theoretical Butler and Poulter (2005)
C. neoformans grubii (serotype A)/Cne PRP8 171,  Theoretical Liu and Yang (2004)
C. neoformans neoformans (serotype D)/Cne 172,  Experimental Butler et al. (2001),
PRP8 Liu and Yang (2004)
Eupenicillium baarnense CBS 134.41/Eba 168,  Experimental Elleuche et al. (2009)
PRP8
E. crustaceum CBS 244.32/Ecr PRP8 158,  Experimental Elleuche et al. (2009)
Histoplasma capsulatum/Hca PRP8 534, 535, + Experimental Liu and Yang (2004),
Butler et al. (2006)
Neosartorya aurata NRRL 4378/Nau PRP8 164,  Theoretical Butler et al. (2006)
N. fenelliae NRRL 5534/Nfe PRP8 155,  Theoretical Butler et al. (2006)
N. fischeri FRR0181/Nfi PRP8 517, + Theoretical Butler et al. (2006)
N. glabra FRR 2163/Ngl-2163 PRP8 155,  Theoretical Butler et al. (2006)
N. glabra FRR 1833/Ngl-FRR1833 PRP8 169,  Theoretical Perler (2002)
N. pseudofischeri FRR 0186/Nps PRP8 169,  Theoretical Butler et al. (2006)
N. quadricincta NRRL 4175/Nqu PRP8 169,  Theoretical Butler et al. (2006)
N. spinosa FRR4595/Nsp PRP8 169,  Theoretical Butler et al. (2006)
Phycomyces blakesleeanus NRRL 198,  Theoretical Perler (2002)
155/Pbl PRP8
Paracoccidioides brasiliensis Pb18/Pbr PRP8 573, + Theoretical Butler and Poulter (2005),
Butler et al. (2006)
Penicillium chrysogenum/Pch PRP8 157,  Experimental Elleuche et al. (2006)
P. expansum/Pex PRP8 162,  Experimental Elleuche et al. (2006)
P. vulpinum/Pvu PRP8 161,  Experimental Elleuche et al. (2006)
Pyrenophora tritici-repentis Pt-1C-BF/Ptr 844, + Theoretical Perler (2002)
PRP8
Spizellomyces punctatus/Spu PRP8 473, + Theoretical Perler (2002)
Uncinocarpus reesii/Ure PRP8 180,  Theoretical Butler et al. (2006)

inteins have been identified in any other species of
the genus Cryptococcus, nor have they been found
in the order Tremellales, nor in the phylum Basi-
diomycota, including Ustilago maydis, Coprinop-
sis cinerea, and Phanerochaete chrysosporium,
species for which the whole genome sequence is
available (Butler et al. 2006).
Allelic PRP8 inteins that occupy the same
insertion site as the Cne PRP8 have, however,
been identified in various species of the phylum
Ascomycota. Particularly, PRP8 inteins were found
in species of the genera Neosartorya, Aspergillus,
and Penicillium of the order Eurotiales. PRP8
inteins of A. fumigatus and A. nidulans are large
inteins and contain an endonuclease domain (Liu
and Yang 2004). The Afu PRP8 intein is an excep-
tionally long PRP8 intein and is comprised of 819
aa (Afu-FRR0163 PRP8) and 820 aa (Afu-Af293
PRP8; Table 3.2). In addition to a 455-aa endonu-
clease domain, a 222-aa sequence is integrated after
block D of the Afu-FRR0163 PRP8 intein. At the
same position, a so-called tongs subdomain of 69
aa is integrated in the Sce VMA1 intein. On this
basis, the 222-aa domain of Afu PRP8 was defined
 Inteins – Selfish Elements in Fungal Genomes
as a putative tongs domain, although there is no
apparent sequence homology between the Afu
PRP8 and the Sce VMA1 domains (Liu and Yang
2004). The only other putative tongs domain was
identified in the large PRP8 intein from Botrytis
cinerea. The domain of Bci PRP8 is at exactly the
same position as the putative tongs domain of A.
fumigatus and shares a high degree of similarity
(Poulter et al. 2007). Overall the tongs domain
seems not to be an essential feature of the intein
function, because the domain is located in a region
of the intein where mutations frequently occur
(Poulter et al. 2007). Also, deletions of selected
amino acids within this region in a homologous
mini-intein from Penicillium chrysogenum did not
result in the complete loss of intein function
(Elleuche et al. 2008).
PRP8 inteins were also identified in species of the genus
Neosartorya, a genus closely related to the genus Aspergil-
lus, in the genera Eupenicillium and Penicillium, as well in
the pathogens Histoplasma capsulatum, Paracoccidioides
brasiliensis, and in its non-pathogenic relative Unicarpus
reesii (Butler et al. 2006; Elleuche et al. 2006; Poulter et al.
2007; Theodoro et al. 2008; Table 3.2).
Phylogenetic analysis based on the internal tran-
scribed spacer sequences (ITS) of Penicillium and
Eupenicillium species revealed that species con-
taining a PRP8 intein are distinct from species
lacking the intein (Elleuche and Pöggeler, unpub-
lished data). Moreover, we and others observed
that intron are often inserted close to the empty
insertion sites in intein-free alleles (Butler et al.
2006; Elleuche and Pöggeler, unpublished data).
In the genus Eupenicillium, all introns are identi-
fied in intein-free prp8 alleles located between 13
base pairs (bp) and 15 bp downstream of the PRP8
intein insertion site (Elleuche et al. 2009). To date,
however, introns at the same insertion site as the
PRP8 intein have not been found in any fungus. In
total, PRP8 inteins were detected in 32 different
fungal species of the phyla ascomycota, basidio-
mycota, chytridiomycota and zygomycota. The
844-aa PRP8 intein of Pyrenophora tritici-repentis
is the largest PRP8 intein described so far. This
intein appears to possess a large insertion after
block C within the endonuclease domain (see
Note at InBase; Perler 2002)). The smallest PRP8
intein of 155 aa was identified in Neosartorya fenel-
liae (Perler 2002). Only 15 out of the 32 fungal
PRP8 inteins (approx. 47%), contain an endonucle-
ase domain, in contrast to the VMA1 inteins of
49
yeasts, where all known inteins contain an endonu-
clease domain (Table 3.1). Of the known inteins of
all three domains of life, only about 15% lack the
endonuclease domain (Perler 2002).
Nearly all PRP8 inteins have clear homology,
based on a high degree of sequence identity and
the position of their insertion within the prp8
gene. The vast majority of fungal PRP8 inteins
occupy the same insertion site as the prototype
allele of Cne PRP8. The PRP8 inteins of only two
chytrid fungi are not homologous and were
identified at two different non-allelic positions:
the PRP8 intein of Spizellomyces punctatus Spu
PRP8 and Bde-JEL423 PRP8-1, one of the two
PRP8 inteins of Batrachochytrium dendrobatidis
(Perler 2002; Table 3.2). Nonetheless, PRP8
inteins are not universal. Various fungal species
have been analyzed to date that do not contain an
intein in the prp8 gene (Elleuche et al. 2006;
Poulter et al. 2007).
2. Activity of Fungal PRP8 Inteins
One of the most interesting questions concerning
inteins is their splicing activity. Since most inteins
are embedded in essential proteins that are often
involved in nucleic acid metabolism, they must be
removed from the precursor protein for the
mature protein to perform its function.
Most experiments on the functionality of fun-
gal inteins were done by expression in heterolo-
gous systems, mainly using Escherichia coli as a
host. To track the splicing reaction, spliced pro-
ducts were usually visualized by SDS-PAGE and
Western blot after overexpression of plasmid-
borne fusion constructs. When inteins are embed-
ded in foreign proteins, they can splice themselves
out, so heterologous extein sequences such as malt-
ose binding protein, GST- or His-tags, and thior-
edoxin often serve as tags. All of these can be easily
detected by Western blot (Wu et al. 1998a; Liu et al.
2003; Liu and Yang 2004; Elleuche et al. 2006). The
yield of spliced products varies with the extein
sequence used, although activity is often higher
when the splicing reaction occurs with native
extein flanks (Xu et al. 1993; Perler 2005).
For the PRP8 inteins, best-characterized is the
splicing of PRP8 mini-inteins from C. neoformans
and from species of Penicillium (Liu and Yang
2004; Elleuche et al. 2006; Pearl et al. 2007a, b).
To characterize the splicing activity of the
50 Skander Elleuche and Stefanie Pöggeler
Cne PRP8, as well as large PRP8 inteins from
A. nidulans, A. fumigatus, and H. capsulatum,
each intein was cloned into a model gene encoding
5 aa of its native extein sequences on each site of
the intein and an N-terminal maltose-binding
protein and a C-terminal thioredoxin tag (Liu
and Yang 2004). After overexpression, SDS-
PAGE, and Western blot analysis with an anti-
thioredoxin antibody, splicing products were
identified. All inteins investigated by Liu and
Yang (2004) were demonstrated to be highly
active when heterologously expressed in E. coli.
The Hca PRP8 intein exhibited a 75% decrease in
splicing activity when shifted from 25 C to 37 C.
This temperature shift is an important physiolog-
ical stimulus for the morphotypic transition from
mold to yeast of H. capsulatum (Woods 2002).
Temperature-independent splicing activity was
reported for three Penicillium PRP8 inteins
(Elleuche et al. 2006).
Another system to characterize PRP8 intein
activity was applied by Pearl et al. (2007a), based
on an assay previously developed for the Myco-
bacterium tuberculosis RecA large intein (Davis
et al. 1992; Buskirk et al. 2004; Skretas and Wood
2005). This method interrupted the a-comple-
mentation peptide of E. coli b-galactosidase,
allowing assessment of intein function in vivo in
E. coli, in combination with immunoblot analysis
of the tagged intein sequences. E. coli loses its
ability to grow on lactose, if the inserted intein is
inactive. The authors examined in detail the influ-
ence of adjacent extein residues by replacing
amino acids 1, 2 and +1, +2 with various
other residues. The results confirmed that splicing
efficiency depends on the composition of extein
sequences (Pearl et al. 2007b). A similar result was
obtained for the Pex PRP8 intein, which was able
to undergo protein splicing within the green
fluorescent protein (GFP). Only the +1 residue
(a Serine) was conserved in the GFP and as
expected, the yield of spliced product was reduced
compared to results obtained with native flanking
extein regions (Elleuche et al. 2006).
To gain further insights into the splicing mech-
anism of fungal inteins, alanine-scanning mutagen-
esis was applied to investigate the influence of single
amino acid residues in Cne PRP8 on protein splic-
ing (Pearl et al. 2007a). Similar to bacterial inteins
and the large Sce VMA1 intein (Southworth et al.
1999; Shingledecker et al. 2000), mutation of
the first, penultimate, and last residues, as well as
mutation in a highly conserved TxxH motif in block
B, strongly inhibited or completely blocked the
splicing reaction. Furthermore, five new residues
were found to be crucial for splicing activity in
this study. All of these residues are located in the
well-conserved block F and have not been previous-
ly determined to be involved in the protein splicing
of bacterial inteins (Pearl et al. 2007a).
As mentioned above, large inteins derived
from bacteria or the VMA1 intein of S. cerevisiae
remain active after the endonuclease domain has
been deleted, indicating that inteins are highly
robust genetic elements. To define the catalytic
and structural elements involved in protein splic-
ing of naturally occurring mini-inteins, an analy-
sis of the structural requirements of protein
splicing has been conducted for the 157-aa Pch
PRP8 intein, which is among the smallest known
nuclear-encoded active splicing protein elements
(Table 3.2). Amino acid sequences of Pch PRP8
can be deleted at two different sites without affect-
ing splicing activity. One site corresponds to the
insertion site of the endonuclease domain in large
allelic PRP8 inteins. The other site was detected at
a new position corresponding to the insertion site
of a putative tongs domain of a large fungal PRP8
intein (see above). The smallest functional intein
found after deleting eight and six amino acids at
two different sites comprises only 143 residues
and is the smallest functional eukaryotic intein
engineered so far (Elleuche et al. 2008). Moreover,
it was demonstrated that the Pch PRP8 intein is
capable of protein splicing in trans (Elleuche and
Pöggeler 2007). After artificially splitting the Pch
PRP8 intein into an N- and a C-terminal domain
at three different sites, protein trans-splicing has
been shown to occur at two sites. One of the
functional split sites corresponds to the insertion
site of the endonuclease domain in allelic large
PRP8 inteins, and the other was detected at a
new position, located N-terminal to the endonu-
clease insertion site (Elleuche and Pöggeler 2007).
C. Other Fungal Inteins
To date, 12 non-allelic sites of intein gene inser-
tion in nine different nuclear genes have been
described in fungi (Perler 2002). In the yeasts
Debaroyomyces hansenii and Candida (Pichia)
guilliermondii, and in the filamentous ascomycete
Podospora anserina, inteins have also been
 Inteins – Selfish Elements in Fungal Genomes
discovered within the glt1 gene encoding gluta-
mate synthase. These belong to the group of large
inteins and are all embedded at exactly the same
site of the GLT1 protein. Recently, a further GLT1
intein was identified in the GLT1-protein in the
plant pathogen Phaeosphaeria nodorum (Poulter
et al. 2007). In addition to the GLT1 intein, P.
anserina contains a non-allelic large intein in the
chitin synthase gene chs2 (Butler et al. 2005). Both
GLT1 and CHS2 inteins contain domains that
indicate the presence of a homing endonuclease
of the LAGLIDADG type. Interestingly, the glt1
gene of A. nidulans contains a currently unanno-
tated intron that occupies the same insertion site
as the Dha GLT1 (Butler et al. 2005).
Phylogenetic analysis revealed that Pan CHS2
is closely related to the GLT1 inteins. Therefore,
Poulter et al. (2007) proposed that Pan CHS2 may
have been derived from an ectopic movement of a
GLT1 intein into a non-allelic CHS2 site. No CHS2
intein has been found in the genomes of any other
fungus: a possible explanation for this might be a
conserved valine residue at the putative +1 extein
position. The CHS2 protein of P. anserina seems to
be the sole known exception with a cysteine at this
position (Butler et al. 2005). A large intein and a
mini-intein have also been detected in the threonyl
tRNA synthetase gene thrRS of the yeast Candida
tropicalis and C. parapsilosis, respectively. The
large Ctr ThrRS intein is 345 aa in length with
some endonuclease-resembling domains, but the
region seems to have accumulated multiple of
mutations that might result in an inactive endonu-
clease. (Poulter et al. 2007). The allelic 182-aa
mini-intein of C. parapsilosis (Cpa ThrRS) has
lost the endonuclease domain (Poulter et al.
2007). Except for mini-inteins of PRP8 proteins,
Cpa ThrRS is the sole example of a eukaryotic
mini-intein.
In prokaryotes, inteins are often embedded in
proteins involved in replication, transcription, or
in related processes such as the metabolism of
nucleotides (Liu 2000).
In general, eukaryotes encode three RNA
polymerases involved in the synthesis of messen-
ger, ribosomal, and transfer RNAs (Cramer et al.
2008). Detailed investigation of eukaryotic RNA
polymerase subunits resulted in the identification
of inteins within this functional category of genes.
The inteins are found in the second largest sub-
unit of either RNA polymerase I, or RNA poly-
merase II, or RNA polymerase III. The insertion
51
sites of inteins in RNA polymerases are among the
most highly conserved regions of these subunits
(Goodwin et al. 2006).
A 456-aa intein is embedded within a subunit of the RNA
polymerase I (RPA2) of the plant pathogen P. nodorum. Pno
RPA2 contains degenerated motifs of an endonuclease do-
main that no longer seems to be active (Goodwin et al.
2006). Three large inteins were also identified within the
gene encoding the second largest subunit of RNA polymer-
ase II (RPB2). Two of them are inserted at the same site in
the zygomycete Spiromyces aspiralis (Sas RPB2-b) and the
chytrid Coelomomyces stegomyiae (Cst RPB2-b). The third
intein, Bde RPB2-c, was identified in another chytridiomy-
cete, B. dendrobatidis, at a non-allelic position. Another
non-allelic RPB2 intein, namely at the a-site, was described
in the RNA polymerase II of the green alga Chlamydomonas
reinhardtii (Goodwin et al. 2006). According to InBase, an
endonuclease-containing intein was recently identified in
the second largest subunit of RNA polymerase III in the
chytrid B. dendrobatidis (Perler 2002). The sequences of the
Bde RPB2 and Bde RPC2, as well as the regions of the
insertion sites, are very similar at the amino acid level.
However, the remaining parts of the exteins are not con-
served, which suggests an intein transfer that recently oc-
curred (InBase; Poulter, Butler and Goodwin, unpublished
data). In contrast to this, the three non-allelic inteins in the
RNA polymerase II are not closely related to each other
(Poulter et al. 2007).
Inteins have been described in four fungal phyla:
Ascomycota, Basidiomycota, Chytridiomycota,
and Zygomycota. Only the PRP8 intein has been
discovered in all fungal phyla. In fungi, inteins are
embedded within nine different genes and with
few exceptions they are inserted at allelic sites
(Tables 3.1–3.3). In eukaryotes, only a few species
are known that contain more than one intein in
their genome. As can be seen from Table 3.1
and Table 3.3, two inteins have been identified in
D. hansenii (Dha VMA1, Dha GLT1), P. anserina
(Pan GLT1, PanCHS2), C. tropicalis (Ctr VMA1,
Ctr ThRS), and P. nodorum (Pno GLT1, Pno
RPA2), and five inteins can be found in
the genome of the chytrid B. dendrobaditis. Pro-
karyotes often contain a variety of inteins in their
genomes, for example, 19 inteins were identified
in the genome of the methanogenic archaeon
Methanococcus jannaschii (Pietrokovski 1998b).
Nucleus-encoded inteins outside the fungi
were recently identified in the green alga C. rein-
hardtii and the slime mold Dictyostelium discoi-
deum (Goodwin et al. 2006). In addition, other
eukaryotic inteins have been described in
plastids from algae and in eukaryotic viruses
52 Skander Elleuche and Stefanie Pöggeler

Table 3.3. Characteristics of other fungal inteins

Species/intein Host Intein size, Activity References

protein endonuclease
Podospora anserina/Pan CHS2 CHS2 649, + Theoretical Butler et al. (2005)
Batrachochytrium dendrobatidis eIF-5B 419, + Theoretical Perler (2002)
JEL423/Bde-JEL423 eIF-5B
Debaryomyces hansenii CBS767/Dha GLT1 GLT1 607, + Theoretical Butler et al. (2005)
Phaeosphaeria nodorum SN15/Pno GLT1 GLT1 635, + Theoretical Poulter et al. (2007)
Pichia (Candida) guilliermondii/Pgu GLT1 GLT1 553, + Theoretical Butler et al. (2005)
Podospora anserina/Pan GLT1 GLT1 682, + Theoretical Butler et al. (2005)
Phaeosphaeria nodorum SN15/Pno RPA2 RPA2 456, + Theoretical Goodwin et al. (2006)
Batrachochytrium dendrobatidis RPB2-c 488, + Theoretical Goodwin et al. (2006)
JEL197/Bde-JEL197 RPB2-c
Coelomomyces stegomyiae/Cst RPB2 RPB2-b 362, + Theoretical Goodwin et al. (2006)
Spiromyces aspiralis NRRL 22631/Sas RPB2 RPB2-b 354, + Theoretical Goodwin et al. (2006)
Batrachochytrium dendrobatidis RPC2 488, + Theoretical Perler (2002)
JEL423/Bde-JEL423 RPC2
Candida parapsilosis CLIB214/Cpa ThrRS ThrRS 182,  Theoretical Poulter et al. (2007)
Candida tropicalis ATCC750/Ctr ThrRS ThrRS 345, + Theoretical Perler (2002)

(Pietrokovski 1998a; Ogata et al. 2005). For exam-
ple, a non-allelic intein was identified in an an-
cient eukaryotic viral DNA, encoding an RNA
polymerase subunit. The viral intein-containing
DNA is embedded in the genome of the oomycete
Phytophthora ramorum (Goodwin et al. 2006).
III. Mobility, Evolution, and
Domestication of Inteins
A variety of inteins can invade DNA sequences by
virtue of the endonuclease encoded within them
(Chevalier and Stoddard 2001). The lateral transfer
of an intervening sequence (either an intron or
intein) into a homologous intron-less/intein-less
allele is termed ‘homing’ and must be distinguished
from the transposition process to non-allelic sites
(Dujon 1989). Phylogenetic analyses have revealed
that the evolutionary biology of mobile inteins is
highly dynamic. Further, VMA1 inteins have been
shown to be the ancestors of the HO endonuclease
required for mating type switching in yeast.
A. Mobility of Fungal Inteins
Intein-homing relies on endonucleases that create
specific double-strand breaks at cognate alleles
that do not contain these mobile elements. Mating
of an intein-less and intein-containing strain leads
to intein transmission to about 75% of the meiotic
progeny. This non-Mendelian inheritance pattern
ensures the persistence of homing endonuclease
genes in populations and leads to their invasion of
new species by horizontal transmission. Intein-
containing genes are not deleterious to their host
organisms because they have evolved in
association with an intein that is removed by pro-
tein splicing (Chevalier and Stoddard 2001; Gogar-
ten et al. 2002). Fungal intein-encoded
endonucleases belong to the LAGLIDADG family
and are characterized by two copies of this con-
served motif. As described in Sect. II.A, the VMA1
intein endonuclease PI-SceI of S. cerevisiae is
among the best-characterized homing endonu-
cleases. PI-SceI initiates the mobility of the intein
by cleaving at intein-less alleles of the VMA1 gene
(Gimble and Thorner 1992). Subsequent to
purification of PI-SceI, genetic and biochemical
studies demonstrated that the endonuclease
makes numerous base-specific and phosphate
backbone contacts with its 31-bp asymmetrical
recognition site (Gimble and Thorner 1993;
Gimble and Wang 1996).
It binds to a 36-bp DNA substrate on intein-free VMA1
alleles and cleaves in a Mg2+- or Mn2+-dependent reaction
to yield 4-bp extensions with 30 overhangs (Gimble and
Wang 1996; Wende et al. 1996; Moure et al. 2002; Noël
et al. 2004). The intein-containing allele is immune to
hydrolysis by PI-SceI, as the VMA1 intein disrupts the
31-bp recognition sequence.
Homing of the Sce VMA1 intein occurs only
during meiosis and not during mitosis, even if
intein-containing and intein-free VMA1 alleles
coexist in the same cell and the VMA1 gene is
 Inteins – Selfish Elements in Fungal Genomes
expressed in mitosis (Gimble and Thorner 1992).
A functional genomic approach revealed that at
least two karyopherins, Srp1p and Kap142p, are
required for the nuclear localization of PI-SceI.
The nuclear localization of PI-SceI was shown to
be induced by inactivation of TOR kinases. More-
over, inactivation of TOR signaling or acquisition
of an extra nuclear localization signal in the
PI-SceI coding region leads to artificial nuclear
localization of the endonuclease and thereby
induces homing even during mitosis. Thus, the
intein-encoded endonuclease utilizes the host sys-
tems of nutrient signal transduction and nucleo-
cytoplasmic transport to ensure the propagation
of its coding region (Nagai et al. 2003). Further-
more, it was demonstrated that the repair of PI-
SceI-induced double-strand breaks occurs at the
same period as that of SPO11-initiated meiotic
recombination and that it is dependent on the
host homologous recombination system as well
as on premeiotic DNA replication (Fukuda et al.
2003, 2004). Several VMA1-derived endonucleases
were shown to fail to cleave their own intein-less
DNA substrate (Posey et al. 2004). Only two
enzymes have been demonstrated to be active:
PI-ZbaI from Zygosaccharomyces bailii and PI-
ScaI from Saccharomyces cariocanus. Sequence
alignment of active site residues revealed that
inactive endonucleases lack one or both of the
conserved acidic residues corresponding to D-
218 and D-326 in PI-SceI (Posey et al. 2004). It is
not known whether any other large fungal intein,
besides the VMA1-derived endonucleases has
an active homing endonuclease. However, the
homing endonuclease domains of all of the PRP8
inteins, except Pbr PRP8 and Cla PRP8, were
demonstrated to have both of the conserved as-
partate residues (Poulter et al. 2007; Theodoro
et al. 2008). Furthermore, an analysis of the fre-
quency of synonymous versus non-synonymous
changes indicated that PRP8-encoded endonu-
cleases have been selectively constrained (Butler
et al. 2006). Together, these data suggest that
many PRP8-derived endonucleases might be ac-
tive. Fungal GLT1-derived endonucleases, except
for Cgu GLT1, also possess the two conserved
aspartate residues, while the first aspartate residue
has been substituted in the Pan CHS2 endonucle-
ase domain. The other fungal inteins (Table 3.3)
do not even exhibit clear evidence of the con-
served motifs C, D, E, and H (Poulter et al.
2007). Thus it seems that only Dha GTL1, Pan
53
GTL1, and Pno GTL1 might contain an active
endonuclease domain (see Sect. II.C).
B. Evolution of Fungal Inteins
Goddard and Burt (1999) formulated a cycle
model consisting of recurrent cycles of intron
invasion, maintenance, degeneration, and loss
for mobile introns with an endonuclease activity.
A modified model was applied to large inteins
(Gogarten et al. 2002; Koufopanou et al. 2002;
Burt and Koufopanou 2004). According to this
model, a mobile intein initially invades a genome
by horizontal transmission. Once situated in the
new genome, it is vertically transmitted to succes-
sive generations and spread out in the population
by homing. When the intein is fixed in the popu-
lation, there is little or no selection against point
or deletion mutations and the activity of the en-
donuclease is destroyed. After the loss of activity,
the endonuclease domain of the intein may even-
tually be deleted. The splicing domain is predicted
to be under a different selection regime. Since the
functionality of the splicing domain is critical for
the function of the host protein, which in most
cases is an essential protein, a strong purifying
selection occurs to preserve function. Only a pre-
cise deletion of the intein-splicing domain will
ensure host protein function and results in a
genome that is devoid of the entire mobile intein.
Before degeneration of the endonuclease, muta-
tion of the endonuclease domain may create an
enzyme that evolves a new specificity and can be
transferred by horizontal transfer to a new host
genome with the recognition site for the altered
enzyme. At this point, the cycle begins again.
Regarding VMA1 inteins of saccharomycete
yeasts, several lines of evidences support the
model of the homing cycle. First, Koufopanou
et al. (2002) and Okuda et al. (2003) reported
that horizontal transmission of VMA1 inteins
from different strains of S. cerevisiae, saccharo-
mycete yeasts, and other yeast species has been a
regular occurrence in their evolutionary history.
Second, the evolutionary state of VMA1-derived
endonucleases from 12 yeast species was
addressed by assaying their endonuclease activ-
ities (Posey et al. 2004).
As stated above, only two enzymes have been shown to be
active. PI-ZbaI cleaves the Z. bailii recognition sequence
54 Skander Elleuche and Stefanie Pöggeler
significantly faster than the S. cerevisiae site, which differs at
six nucleotide positions. A mutational analysis indicated
that PI-ZbaI cleaves the S. cerevisiae substrate poorly due
to the absence of a DNA-protein contact that is established
by PI-SceI. These findings demonstrated that intein homing
endonucleases evolve altered specificities as they adapt to
recognize alternative target sites (Posey et al. 2004).
Finally, in the tetraploid wild-wine S. cerevisiae
strain DH1-1A, it was shown that an intein-
containing VMA1 allele and an intein-less VMA1
allele are encoded in one genome. The molecular
reason for the co-existence of both alleles in one
genome was shown to be due to a loss of activity in
the PI-SceI analogue encoded by the DH1-1A
VMA1 intein, and mutations in the 31-bp recogni-
tion site of the intein-free allele of DH1-1A
(Gimble 2001). In contrast, recent analyses of the
PRP8 inteins suggest that the homing cycle model
is not generally applicable (Gogarten and Hilario
2006). Compared to the endonucleases of VMA1
inteins, the homing endonucleases of PRP8 inteins
show high dS/dN values (Butler et al. 2006).
A high value of the quotient of the frequency of synony-
mous (dS) versus the frequency of non-synonymous
changes (dN) implies that the endonuclease is functional
(Butler et al. 2006).
In addition, the level of synonymous change in the
PRP8 endonuclease has reached saturation while
the VMA1 endonuclease is not saturated by synon-
ymous substitutions. These results imply that
VMA1 inteins are of more recent origin than the
PRP8 inteins. Furthermore, phylogeny of the euas-
comycete PRP8 inteins provides no evidence for
horizontal transfer. Only basidiomycetes of the
genus Cryptococcus may have gained the PRP8
intein by horizontal transmission (Butler and Poul-
ter 2005; Butler et al. 2006). Based on these results,
Butler et al. (2006) proposed a modified model for
the evolution of inteins present in euascomycetes.
They suggested that inteins might be maintained by
balancing selection. They may not become fixed in a
population due to a decreased fitness of the host
organism, and thus, the homing cycle might operate
in sub-populations only. This scenario would allow
continuous selection for the endonuclease function
during extended periods of vertical transmission.
C. Domestication of Fungal Inteins
Per se, it cannot be ruled out that inteins contrib-
ute to host fitness rather than being detrimental.
It has been reported that PI-SceI binds but does
not cut the promoter of the S. cerevisiae GSH11
gene encoding a high-affinity glutathione trans-
porter. GSH11 is not expressed in a PI-SceI-
deleted strain, and the inability to express GSH11
has been shown to be overcome by the introduc-
tion of the coding region of PI-SceI or the entire
VMA1 gene (Miyake et al. 2003). Another example
for the domestication of an intein is the HO endo-
nuclease, required for mating type switching in
various yeast species (Haber 1998). Phylogenetic
analyses revealed that HO and the VMA1 intein of
S. cerevisiae are close relatives (Dalgaard et al.
1997). The HO gene is thought to have arisen by
an ectopic insertion of the coding region of the
VMA1 intein (Butler et al. 2004). The HO endonu-
clease is encoded by a free-standing gene showing
50% similarity to the full-length VMA1 intein,
including the splicing domain, but is unique
among LAGLIDADG endonucleases in having a
120-residue C-terminal putative zinc finger do-
main. Mutational analysis indicated that in addi-
tion to the splicing domain and the zinc finger
domain, conserved residues between HO and cat-
alytic, active-site residues in PI-SceI, and other
related homing endonucleases are essential for
HO activity (Bakhrat et al. 2004). Phylogenetic
analysis from several yeast species revealed a
single origin of the HO gene from the VMA1
intein. In contrast to VMA1 inteins, HO shows
no evidence for degeneration or horizontal trans-
mission (Koufopanou and Burt 2004; Bakhrat
et al. 2006).
IV. Application of Inteins
In recent years, the utilization of inteins has be-
come a major focus in biotechnological research.
This section focus on and briefly reviews various
intein-related applications for the analysis of pro-
tein–protein interactions, regulation of protein
activity, and protein purification.
A. Inteins and Their Application in Protein–
Protein Interaction Studies
Protein–protein interactions play key roles in
various processes of biological systems, including
transcriptional regulation, protein sorting,
and receptor-ligand interactions. A number of
 Inteins – Selfish Elements in Fungal Genomes
techniques for the detection of protein–protein
interactions, such as yeast two-hybrid, split-ubi-
quitin, bimolecular fluorescence complementa-
tion, or fluorescence detected resonance energy
transfer have been invented and established in
recent years (Fields and Song 1989; Rossi et al.
1997; Hink et al. 2002; Pasch et al. 2005). Several of
these methods are often used in fungi, e.g. to
detect or prove the interaction of fungal proteins
(Hoff and Kück 2005; Nolting and Pöggeler 2006).
All strategies used must result in the detection of a
specific protein–protein interaction via conver-
sion to a traceable signal. An intein-based method
to study in vivo protein protein interactions in a
mammalian system, as well as in bacteria, has
been published by Ozawa et al. (2000, 2001). The
technique relies on the reconstitution of a split
enhanced green fluorescent protein (GFP) by
intein-mediated protein splicing. Each part of
the GFP is fused as an extein sequence to an intein
moiety derived from an artificially split version of
the Sce VMA1 intein. Neither intein construct can
self-assemble, but interacting proteins fused to the
other sides of the split intein are able to bring the
intein halves in close proximity. This results in
protein splicing and the release of a functional
green fluorescent protein (GFP). The fluorescence
intensity increases proportionally with the num-
ber of interacting protein pairs (Ozawa and Ume-
zawa 2001). A great advantage of this system is the
feasibility to measure protein–protein interac-
tions anywhere in the cell because the method
does not require that the interaction takes place
in a special cell compartment, e.g. in the nucleus,
which is required in the yeast two-hybrid system.
Another genetic approach, using the reconsti-
tution of a split-enhanced green fluorescent pro-
tein (EGFP) by splicing a split DnaE intein derived
from Synechocystis sp. PCC6803, was developed to
investigate cellular localization of proteins. In this
approach, a fusion protein comprised of the IC
moiety of the naturally split Ssp DnaE intein was
fused to one half of the GFP protein and to a
mitochondrial targeting signal. After transforma-
tion, the fusion protein was demonstrated to be
translocated into the mitochondrial matrix. The
other half of the GFP was fused with the IN domain
of Ssp DnaE and this construct was then fused to
cDNA libraries. If a test protein contains a func-
tional mitochondrial targeting signal, it translo-
cates into the mitochondrial matrix, where EGFP
is then formed by protein splicing. The cells
55
harboring this reconstituted EGFP can be
screened rapidly by fluorescence-activated cell
sorting, and the cDNAs can be subsequently
isolated and identified from the cells (Ozawa
et al. 2003). This system has been also elaborated
to identify proteins targeted to the endoplasmic
reticulum (Ozawa et al. 2005). Although not all of
these methods have been tested in fungal systems,
they demonstrate interesting approaches to char-
acterize protein–protein interactions and protein
sorting in the living fungal cell.
B. Regulation of Protein-Splicing Activity
A major aim of physiologists is to temporally
control protein function in living organisms. To
accomplish this aim, the GFP-based system
to study protein–protein interactions was further
elaborated to induce splicing in living cells at a
defined time-point. First, the protein of interest
must be divided into two parts and fused to
inteins, like GFP in the above-mentioned fluores-
cent-based systems. To keep the target protein
inactive, until it is specifically activated in the
cell, both fragments of the protein including the
intein halves are fused to other proteins that in-
teract or dimerize only under certain inducible
conditions. In one approach, two mammalian
proteins (FKBP, FK506-binding protein; FRB,
the binding domain of the FKBP-rapamycin-
associated protein), which are known to bind to
rapamycin were fused to the artificially split Sce
VMA1 intein (Mootz and Muir 2002). Upon addi-
tion of rapamycin, the trans-splicing reaction can
be triggered in vivo or in vitro, and the intein
connects the halves of the desired protein with a
peptide bond (Mootz et al. 2003; Schwartz et al.
2003). This impressive system was termed the
conditional protein splicing (CPS) system and
was used to induce luciferase function in cultured
cells and multicellular organisms. Drosophila mel-
anogaster strains transformed with CPS con-
structs were fed with food containing rapamycin,
resulting in the detection of luminescence in the
living flies (Perler 2007; Schwartz et al. 2007).
Since rapamycin is fairly toxic to yeast, a light-
dependent CPS system was recently developed for
the yeast S. cerevisiae that depends on regulation
of the photo-dimerization of phytochrome B
and the phytochrome interacting factor 3 of Arabi-
dopsis thaliana (Tyszkiewicz and Muir 2008).
56 Skander Elleuche and Stefanie Pöggeler
Dimerization of the plant phytochrome B and the
phytochrome interacting factor 3 occurs in red light
(660 nm) and reversal of the dimerization in far-red
light (750 nm). Thus, a change of the excitation
wavelength to the far-red spectrum (750 nm) results
in the dissociation of interaction partners. Fusion of
phytochrome B and the phytochrome interacting
factor 3 to the artificially split Sce VMA1 intein, and
using the maltose-binding protein (MBP) and a
Flag-tag as exteins, enabled Tyszkiewicz und Muir
(2008) to demonstrate protein splicing under light-
inducing conditions in living yeast cells.
C. Intein-Mediated Protein Purification
To produce recombinant proteins in large
amounts and to purify them from crude extracts
has become a major task for biotechnologists in
recent years. Proteins can be produced in bacte-
rial expression systems and the purification pro-
cess can be eased by the utilization of affinity tags
(Terpe 2003). Different small peptide tags (e.g.
poly-His-, poly-Arg-, CBD-, or S-tags) are com-
monly used, as well as higher molecular weight
tags (e.g. GST- or MBP-tags) to induce the solu-
bility of proteins. However, removal of the affini-
ty tag is often a complicated challenge and
accompanied by different problems. Intein-
mediated protein purification systems attempt
to avoid these problems. In principle, a target
protein is fused at either the N- or the C-terminus
to a modified intein. The other site of the intein is
linked to an affinity tag and is mutated at the tag-
linked splice junction to undergo inducible spe-
cific cleavage with the site on the protein of
interest. Crude protein extracts are loaded on
an affinity column, the tag immobilizes the intein
fusion construct, and after some washing steps,
an induced splicing reaction (cleavage step)
releases the desired protein from the column.
Normally, the cleavage step is inducible under
mild conditions, e.g. by the addition of thiolic
agents, changes in pH or temperature. The first
intein engineered for protein purification was the
VMA1 intein from S. cerevisiae (Chong et al.
1997). Later, naturally occurring or artificially
engineered mini-inteins were predominantly
used for protein purification systems (South-
worth et al. 1999; Wood et al. 1999; Ding
et al. 2007).
Further development in intein-mediated pro-
tein purification was achieved using naturally or
artificially split inteins (Mills et al. 1998; Wu et al.
1998b). The use of these trans-cleavage systems
advanced the purification, because it prevented
premature in vivo cleavage, a problem often ob-
served when cis-splicing inteins are used. The
trans-splicing system takes advantage of the abili-
ty of the intein halves to self-assemble. In the
majority of cases, the Ssp DnaE intein is used
(Chong and Xu 2005). The target protein is fused
to the IN domain of Ssp DnaE, while the IC domain
is mutated at the C-extein cleavage site. Further-
more, both intein halves are tagged to facilitate
isolation and purification of the expressed intein
fusion proteins. Co-incubation of both parts
results in the reconstitution of the intein splicing
activity and in the release of the target protein.
D. Screening Systems for Protein-Splicing
Inhibitors
Using inteins to identify inhibitory agents to
prevent fungal growth is a new field in fungal
biology. Because inteins are localized inside
essential genes, they have been often confirmed
as useful drug targets for preventing fungal infec-
tions (e.g. C. neoformans, A. fumigatus, or
H. capsulatum; Liu and Yang 2004). Thus, assays
were developed to identify new protein-splicing
inhibitors. The above-mentioned split GFP re-
porter system (Ozawa et al. 2000) was applied
for the in vitro screening of antimicrobial agents
inhibiting the splicing reaction of the Mycobac-
terium tuberculosis RecA intein (Gangopadhyay
et al. 2003a, b). The ORF of GFP was disrupted by
integration of Mtu RecA. Initially, the GFP-intein
fusion protein is overexpressed, but remains in-
active because inclusion bodies are formed. After
release from the inclusion bodies and renaturat-
ing of the fusion product, in the absence of an
inhibitor the intein undergoes protein splicing.
When an inhibitor is present, protein splicing
and the emission of fluorescence is prevented
(Gangopadhyay et al. 2003a). The system de-
scribed has already been used to test the fungal
Pex PRP8 intein for its splicing activity within a
foreign host protein and may be used in the
future to design an efficient screening system
for protein-splicing inhibitors of other fungal
inteins (Elleuche et al. 2006).
 Inteins – Selfish Elements in Fungal Genomes
V. Conclusions
Intein are internal protein domains found inside
the coding region of different proteins. They are
transcribed and translated together with their host
protein and are removed from the unprocessed
protein by protein splicing. Fungi encode large
inteins comprising independent protein-splicing
and endonuclease domains, and mini-inteins
which lack the central endonuclease domain. Our
knowledge on the distribution, evolution, and func-
tionality of fungal inteins has expanded enormously
during the past ten years. Currently, more than 70
inteins have been identified within the nuclear gen-
omes of fungi. Most of them are embedded in
homologs of the S. cerevisiae VMA1 gene or within
the prp8 gene, but they can be also found in gluta-
mate synthases, chitin synthases, threonyl-tRNA
synthetases, and subunits of DNA-directed RNA
polymerases. Genomic sequencing projects for sev-
eral fungal species have been completed and many
more are under way (http://www.fgsc.net/). This
will accelerate the discovery of new fungal inteins.
Biochemical and phylogenetic analysis will contrib-
ute further insight into the splicing mechanism and
the evolution of fungal inteins. Ultimately, this will
lead to the engineering of fungal inteins for a variety
of applications.
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4 Apoptosis in Fungal Development and Ageing

DIANA BRUST1, ANDREA HAMANN1, HEINZ D. OSIEWACZ1

CONTENTS plasma membrane results in activation of the ex-

I.
General Description of Apoptosis . . . . . . . . . . . . . . . 63 trinsic signalling pathway. After a death ligand is
A. Apoptosis in Mammals . . . . . . . . . . . . . . . . . . . . . . 63 bound to a specific receptor, initiator caspase-
B. Apoptosis in Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 8 becomes activated and transmits the apoptotic
C. Differences Between Fungal and signal by cleavage of effector caspase-3 (reviewed
Mammalian Apoptosis . . . . . . . . . . . . . . . . . . . . . . . 66
II. Apoptosis in Fungal Development . . . . . . . . . . . . . . . 66 by Walczak and Krammer 2000). Subsequently,
A. Apoptosis in Host–Pathogen and activated caspase-3 leads to the cleavage of specif-
Antagonistic Interactions . . . . . . . . . . . . . . . . . . . . 66 ic substrates, including the inhibitor of the cas-
B. Apoptosis During Fungal Reproduction . . . 69 pase-activated DNase (ICAD) in the cytoplasm
C. The Role of Apoptosis in Fungal and the poly(ADP)ribose-polymerase (PARP) in
Lifespan Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
III. Concluding Remarks and the nucleus (reviewed by Degterev et al. 2003).
Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 The intrinsic pathway of apoptosis is closely
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 connected to mitochondria. Molecular damage of
these organelles leads to mitochondrial outer
membrane permeability (MOMP), which facili-
tates the release of apoptotic factors which are
I. General Description of Apoptosis normally located in the intermembrane space.
Two basic mechanisms inducing MOMP have
A. Apoptosis in Mammals been described. First, pore formation via the per-
meability transition pore complex (PTPC), a com-
The term apoptosis, which originates from the plex constituted by outer and inner membrane
ancient Greek word describing the fall of leaves proteins as well as proteins in the matrix. Primarily
from trees, was first suggested by Kerr et al. (1972) the complex is formed by the voltage-dependent
to describe the process of physiological cell death anion channel (VDAC), also termed porin, in the
in liver cells. Later, it was more generally used as a outer mitochondrial membrane, the adenine nucle-
functional definition of one type of programmed otide transporter (ANT) in the inner membrane
cell death (PCD). The process is characterised by and cyclophilin D in the mitochondrial matrix,
specific morphological features including cell which allows water and solutes up to 1.5 kDa to
shrinkage, chromatin condensation and mem- cross through the outer and inner mitochondrial
brane blebbing, ultimately leading to phagocytosis membrane. PTPC opening is highly sensitive to
of affected cells (reviewed by Hengartner 2000). calcium ions and pro-oxidant agents. The opening
The induction of apoptosis depends on specific results in mitochondrial swelling followed by
signals from the extracellular (extrinsic) or the rupture of the outer mitochondrial membrane.
intracellular (intrinsic) micro-environment (Fig. 4.1). Second, MOMP can result from the formation of
Stimulation of death receptors of the tumor pores in the outer membrane by members of the
necrosis factor (TNF) receptor superfamily in the BAX protein family which integrate into the mito-
chondrial outer membrane promoted by a plethora
of apoptotic stimuli (reviewed by Kroemer et al.
2007).
1
Institute for Molecular Biosciences, Department of Biosciences and
Cluster of Excellence Macromolecular Complexes, Goethe-Univer-
sity, Max-von-Laue-Strasse 9, 60438 Frankfurt, Germany; e-mail: The mammalian protein B-cell lymphoma 2 (BCL-2) is
Osiewacz@bio.uni-frankfurt.de the prototype of a protein superfamily with over 20

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
64
death ligand
EXTRINSIC
PATHWAY death receptor
cytosol
initiator
caspase-8 BID
Apaf-1
apoptosome
ent
nd
pe
de
a se- hway
p t
cas pa
effector
caspase-3 ICAD CAD
(metacaspases)
Fig. 4.1. Schematic representation of apoptotic signalling
pathways in mammals. The extrinsic pathway is charac-
terised by death receptors. After binding of death ligands,
initiator caspase-8 becomes activated resulting in the cleav-
age of effector caspase-3 (reviewed by Walczak and Kram-
mer 2000). Activated caspase-3 is able to cleave specific
substrates like ICAD (inhibitor of the caspase-activated
DNase, CAD) in the cytoplasm and the poly(ADP)ribose-
polymerase (PARP) in the nucleus (reviewed by Degterev
et al. 2003) triggering DNA damage. The intrinsic pathway
depends on the release of apoptotic factors from mitochon-
dria by formation of BAX/BAK pores or permeability transi-
tion pore complexes (PTPC) (reviewed by Kroemer et al.
2007). Liberated AIF and endoG translocate to the nucleus
and execute the caspase-dependent pathway of apoptosis
demonstrating common apoptotic markers like DNA
fragmentation and chromatin condensation. The caspase-
relatives. Members of this family share at least one con-
served BCL-2 homology (BH) domain. BCL-2 related
proteins are divided into three subfamilies due to the
number of their BH domains: (i) members of the BCL-
2 family possess four BH domains, (ii) those of the BAX
family three BH domains and (iii) the proteins of the
BH3-only family contain only the shortest of the four
BH motifs. While members of the BCL-2 family display
an anti-apoptotic activity, proteins of the BAX and BH3-
only family are pro-apoptotic. BH3-only proteins seem to
act as direct antagonists of their anti-apoptotic homolo-
gues, whereas the integration of BAX proteins into the
outer mitochondrial membrane leads to MOMP
(reviewed by Cory and Adams 2002).
Diana Brust et al.
stress
INTRINSIC
PATHWAYS
tBID
mitochondrion
BAX/ PTPC
cyt c BAK
nt
Omi/ Smac/ de
en
caspase-9 ep
ind ay
endoG
Htr2A DIABLO e-
as thw
casp pa AIF
IAP
PARP
nucleus
dependent pathway is characterised by released cytochrome
c (cyt c) which is involved in the formation of the apopto-
some consisting of Apaf-1, caspase-9 and cyt c. The apopto-
some has the ability to activate effector caspase-3 initiating
apoptosis as described before. Smac/DIABLO and Omi/
Htr2A also released from mitochondria neutralise the nega-
tive effects of IAP (inhibitor of apoptosis proteins) on cas-
pase-3 activity in the cytosol (reviewed by Saelens et al.
2004). The extrinsic and intrinsic pathways are linked by
BID. Once BID is cleaved by initiator caspase-8, the cleavage
product tBID supports the liberation of apoptosis proteins
from mitochondria (reviewed by Cory and Adams 2002).
Several factors of different apoptosis pathways also found
in fungi are boxed. The dashed box of BAX/BAK implies that
no structure homologues were found in fungi but heterolo-
gous expression of the mammalian proteins affects the cells
in a pro-apoptotic way as well (e.g. Jürgensmeier et al. 1997)
Once MOMP is triggered, pro-apoptotic mole-
cules migrate from mitochondria into the cytosol,
inducing two different signalling pathways: a cas-
pase-dependent and a caspase-independent path-
way. The main mediator of the caspase-dependent
pathway is cytochrome c which directly initiates
the activation of caspase-3 after leaving the mito-
chondrial intermembrane space via the formation
of the apoptosome complex. Smac/DIABLO and
Omi/Htr2A, two proteins which are also released
from mitochondria, neutralise the negative effects
of IAP (inhibitor of apoptosis proteins) on
 Apoptosis in Fungal Development and Ageing
caspase-3 activity in the cytosol (reviewed by
Saelens et al. 2004). The receptor and the mito-
chondrial pathway are connected via BID, a mem-
ber of the BH3-only family (reviewed by Cory and
Adams 2002). Activated caspase-8 of the extrinsic
pathway is able to cleave BID, leading to the gen-
eration of tBID which subsequently migrates to
mitochondria and causes cytochrome c release.
The caspase-independent pathway is executed
after the liberation of apoptosis-inducing factor
(AIF) and endonuclease G (endoG) from mito-
chondria (reviewed by Saelens et al. 2004). After
translocation of these proteins to the nucleus they
cause DNA fragmentation and chromatin conden-
sation, two typical apoptosis markers in mammals.
B. Apoptosis in Fungi
More than ten years ago the first evidence of a
molecular apoptotic machinery emerged in Sac-
charomyces cerevisiae when Madeo et al. (1997)
described typical apoptotic markers like chroma-
tin condensation and DNA fragmentation. Subse-
quently, evidence for apoptosis was also reported
in other fungi. For instance, in Aspergillus fumi-
gatus the typical apoptotic DNA laddering was
noticed after the application of stress to growing
cultures (Mousavi and Robson 2004). The TUNEL
assay was successfully applied to visualise
DNA strand breaks in different fungal systems.
For example, in yeast DNA breaks were demon-
strated in a mutant defective in cell cycle (Madeo
et al. 1997), in A. fumigatus protoplasts from
mycelia entering the stationary phase tested
TUNEL-positive (Mousavi and Robson 2003),
and the TUNEL assay was also successfully
applied in hyphae of Colletotrichum trifolii
(Chen and Dickman 2005).
Furthermore, another apoptotic characteristic
of mammalian cells, the relocalisation of phospha-
tidylserine (PS) from the inner to the outer layer
of the plasma membrane was demonstrated by
annexin V staining in S. cerevisiae (Madeo et al.
1997) and in several filamentous fungi (e.g.
Hamann et al. 2007; Richie et al. 2007).
One of the most common apoptotic features is
clearly the release of cytochrome c from mito-
chondria to trigger the activation of caspases, the
main players of mammalian apoptosis. Until now,
translocation of cytochrome c to the cytosol could
65
only be demonstrated in yeast cells after treatment
with acetic acid (Ludovico et al. 2002) but not in
filamentous fungi. Silva et al. (2005) reported that
deletion strains of cytochrome c are more resis-
tant against hyperosmotic stress than the wild
type, supporting the idea that cytochrome c plays
a role in fungal apoptosis. Significantly, although
cytochrome c release has not been demonstrated
yet in filamentous fungi, several reports suggest
caspase-like activity in total protein extracts from
different fungi using available mammalian cas-
pase substrates (Madeo et al. 2002; Thrane et al.
2004; Richie et al. 2007). Interestingly, contempo-
rary degradation of PARP was observed in the
presence of fungal protein extracts containing
high caspase-like activity (Thrane et al. 2004).
Specific proteases, termed metacaspases and shar-
ing partial sequence homology with mammalian
caspases, were suggested to be responsible for
caspase-like activity determined in fungal cell
extracts. However, today there is strong evidence
for differences in the cleavage site in metacaspases
and mammalian caspases (reviewed by Vachova
and Palkova 2007). While caspases cleave aspar-
tate-containing peptides, optimal cleavage activity
of some metacaspases was detected using sub-
strates with an arginine residue on P1 position of
the peptide substrate (Gonzalez et al. 2007;
Hamann et al. 2007).
In addition to a (meta)caspase-dependent path-
way, a caspase-independent pathway of apoptosis
appears to be active in fungi. Generally, in yeast AIF
and endoG homologues were identified and char-
acterised, indicating the same function of these
proteins as in humans (Wissing et al. 2004; Büttner
et al. 2007). Finally, it has been found that compo-
nents of PTPC are well conserved in eukaryotes.
Surprisingly, a yeast porin deletion strain was
found to be more sensitive to acetic acid than the
wild type. In contrast, deletion of the three ANT-
encoding genes of S. cerevisiae resulted in a higher
stress resistance, while deletion of the yeast cyclo-
philin D encoding gene displayed no effect on acetic
acid-induced apoptosis (Pereira et al. 2007). How-
ever, the yeast cyclophilin D homologue seems to
mediate the copper-induced apoptotic programme
(Liang and Zhou 2007). Interestingly, in spite of the
fact that fungi seem to lack sequence homologues of
the BCL-2 family, heterologous expression in yeast
of genes encoding the mammalian proteins let
to pro- and anti-apoptotic effects, respectively
(Jürgensmeier et al. 1997; Manon et al. 1997). It is
66 Diana Brust et al.
thus possible that fungi possess functional homo-
logues of the BCL-2 superfamily.
C. Differences Between Fungal
and Mammalian Apoptosis
Although mammals and fungi share several apo-
ptotic factors demonstrating programmed cell
death, in lower eukaryotes the signalling pathways
appear to be much simpler than in mammals
(Fig. 4.1). Until now, the characteristic death
receptors triggering the extrinsic pathway of
mammalian apoptosis have not been identified
in fungi. At most, mating pheromones might be
considered as exterior signals recognized by spe-
cific G protein-coupled receptors which normally
activate a well defined MAP kinase signalling cas-
cade (Schrick et al. 1997). High pheromone con-
centrations lead to apoptotic cell death (Severin
and Hyman 2002). In contrast, intrinsic pathways
of apoptosis are clearly active in fungi, but in a
simplified version when compared to mammals.
Although a release of cytochrome c was at least
demonstrated in yeast, the formation of the apop-
tosome could not be detected. In fungi metacas-
pases cleaving proteins at a site differing from
those of mammalian caspases are part of the cas-
pase-dependent intrinsic pathway (reviewed by
Vachova and Palkova 2007). Interestingly, until
now no more than two metacaspase genes could
be identified in fungal genomes, while humans
contain a set of caspases with different tasks
involved in apoptotic and inflammatory processes
(reviewed by Degterev et al. 2003). Similar to
mammals, yeast contains an IAP homologue,
called BIR1, which interacts with the Omi/Htr2A
homologue NMA111 (Fahrenkrog et al. 2004;
Walter et al. 2006). However, mammalian Omi/
Htr2A is normally localised in mitochondria,
while NMA111 is a nuclear protein, further stres-
sing that mammalian and fungal apoptosis clearly
differ in mechanistic details.
The caspase-independent pathways of apo-
ptosis are poorly investigated in fungi. Only in
yeast was evidence shown for AIF- and endoG-
dependent cell death (reviewed by Liang et al.
2008). In the fungal kingdom no structural homo-
logues of human BCL-2 proteins which play an
important role in a pro- and anti- apoptotic man-
ner could be detected, although this human pro-
tein class is active in yeast and other fungi
(reviewed by Eisenberg et al. 2007). Taken togeth-
er, it is tempting to speculate about the rudimen-
tary apoptosis signalling pathways of fungi as an
evolutionary precursor of mammalian apoptosis.
But why and in which context did an apoptotic
programme evolve at this early stage of evolution?
II. Apoptosis in Fungal Development
A. Apoptosis in Host–Pathogen
and Antagonistic Interactions
The observations described above demonstrate
that apoptotic cell death is an evolutionarily con-
served programme which is a common feature of
both higher and lower eukaryotes. Hallmarks of
apoptosis can even be observed in prokaryotes.
This kind of cell death is therefore suggested to be
a consequence of mitochondrial endosymbiosis
and additional horizontal gene transfer events
(Koonin and Aravind 2002). It is suggestive to
assume that the ability to undergo apoptosis pro-
vides some selective advantage. Indeed, some ex-
perimental data point to vital roles of fungal
apoptotic pathways. One example is a competition
experiment in which a selective advantage of
S. cerevisiae cells capable of metacaspase 1-dependent
apoptosis was described (Herker et al. 2004). In a
mixture of wild-type and Yca1-deletion cells, cells
of the deletion strain were outgrown during long-
term cultivation by wild-type cells, suggesting that
wild-type cells dying via apoptosis stimulate the
survival of other wild-type cells. Alternatively, ap-
optotic clearance of aged or harmed cells leads to
a fitter, better adapted population in the long run.
Moreover, during the past ten years, pro-
grammed cell death and especially apoptosis has
been identified to play an important role in pro-
cesses induced via the attack of a host by a poten-
tial fungal pathogen. One of the first host
responses is the so-called oxidative burst, the pro-
duction of high levels of reactive oxygen species
(ROS).
The oxidative burst (also known as respiratory burst in
phagocytes of mammals) is defined as rapid production of
high levels of ROS in response to external stimuli. It
provides the first line of defence against pathogen attack.
Upon recognition of a microbial pathogen, a plant or an
animal host starts to release ROS. These ROS (mostly
superoxide anion and hydrogen peroxide) are produced
by different host enzyme systems. For instance, NADPH
 Apoptosis in Fungal Development and Ageing 67

oxidases produce superoxide anions, which are converted
to hydrogen peroxide by superoxide dismutases (SODs).
Hydrogen peroxide can serve as precursor for hydroxyl
radical generation via Fenton chemistry. The different
types of ROS are able to oxidize proteins, lipids and
DNA. Beside the role of directly attacking pathogens, it is
clear that ROS are also important signalling molecules
inducing several host pathways (Forman and Torres
2002; Gwinn and Vallyathan 2006).
Experimental data suggest that the oxidative burst
might induce apoptosis in the pathogen and/or in
the host. Animal pathogens often target and sup-
press the host’s cell death pathways with different
compounds to successfully establish infection
(Table 4.1). The induction of programmed cell
death is a critical step in the development of a
successful infection and it is thus not surprising
that the triggering of apoptosis is highly relevant
for both host and pathogen.
One example for the role of apoptosis in the
interaction of a pathogen with its host is the infec-
tion of humans by A. fumigatus. An early response
to fungal attack is the induction of an oxidative
burst directed against the germinating spores. Ex-
perimental data suggest that an apoptotic pathway
is induced in this interaction. A. fumigatus cul-
tures treated with 0.1 mM hydrogen peroxide dis-
play the classic hallmarks of apoptosis, including
PS externalisation and DNA fragmentation
(Mousavi and Robson 2004). This treatment
might mimic the physiological scenario in the
pathogen. Comparable ROS levels were measured
during oxidative bursts in Arabidopsis thaliana,
revealing maximal levels of 0.025 mM hydrogen
peroxide (Davies et al. 2006). The induction of
apoptosis by hydrogen peroxide in Aspergillus
fumigatus can completely be blocked by the addi-
tion of the protein synthesis inhibitor cyclohexi-
mide demonstrating dependency of this type of
PCD on de novo protein synthesis.
It seems that not only does the host use apo-
ptosis to get rid of the pathogen, but also the
pathogen utilizes apoptosis to escape being killed
by immune cells. Gliotoxin, a non-ribosomal pep-
tide produced by A. fumigatus has been demon-
strated to display apoptotic properties (Pahl et al.
1996; Suen et al. 2001; Kweon et al. 2003). The
supernatant of A. fumigatus cultures, in which
gliZ (encoding a transcriptional regulator of glio-
toxin production) was deleted, was found to have
a significantly lower ability to induce apoptotic
cell death in polymorphonuclear leukocytes than
wild-type supernatant (Bok et al. 2006). Obvious-
ly, successful establishment of infection in animal
hosts depends strongly on the ability of the path-
ogen to escape killing by the reactive oxygen spe-
cies released by immune cells during the oxidative
burst. Moreover, to support the infection, the
pathogen might profit via the ability to induce
apoptosis specifically in those cells that are able
to kill him, like e.g. neutrophils. The outcome of
an infection event thus clearly depends on the

Table 4.1. Examples of apoptosis-inducing/inhibiting compounds implicated in interactions of fungi with other species

Compounds Type of interaction Putative biological function References

Acetic acid Antagonistic (acetic acid secreting Selective advantage, induces Ludovico et al. (2002)
bacteria vs yeast) apoptotic phenotype
Farnesol Antagonistic (Candida albicans vs Selective advantage, induces Semighini et al. (2006a,
Aspergillus nidulans and Fusarium apoptotic phenotype 2008)
graminearum)
Gliotoxin Host (human)/pathogen (A. fumigatus) Pathogen attack to eliminate Pahl et al. (1996),
immune response Suen et al. (2001),
Kweon et al. (2003)
Killer toxins Antagonistic (different yeast species vs
Selective advantage, induces Klassen and Meinhardt
other yeast species) apoptotic phenotype in (2005), Reiter et al.
susceptible yeasts (2005)
PAF Antagonistic (Penicillium chrysogenum Selective advantage, induces Leiter et al. (2005)
vs A. nidulans) apoptotic phenotype
ROS Host (human)/pathogen (A. fumigatus) Respiratory burst, host defence Mousavi and Robson
mechanism (2004)
a-Tomatine Host (tomato)/pathogen (F. oxysporum) Host defence, induces apoptosis Ito et al. (2007)
in the pathogen
Transferrin Host (Protaetia brevitarsis)/pathogen Prevents host from pathogen- Kim et al. (2008)
(Beauveria bassiana) induced apoptosis
68 Diana Brust et al.
balance between apoptosis resistance and the abil-
ity to induce apoptosis.
Another example of a pathogenic fungus able
to induce apoptotic cell death in the animal host is
Beauveria bassiana, a common and serious insect
pathogen. Recent studies with the white-spotted
flower chafer (Protaetia brevitarsis) revealed an
important role of the insect transferrin protein
in apoptosis prevention. Insect larvae with re-
duced transferrin levels displayed a remarked in-
crease in apoptosis induction upon exposure to
different stressors, like heat stress and hydrogen
peroxide stress, but also to fungal challenge (Kim
et al. 2008). These data again support the idea that
prevention of apoptosis is directly linked to host
survival.
In plant/pathogen interactions the role of ap-
optosis is different. In plants attacked by a patho-
gen in principle two different scenarios can be
observed. In one scenario, in plants which are
able to recognise substances elicited during the
pathogen attack, a defence reaction is induced,
the so-called hypersensitive response, leading to
the induction of programmed cell death in the
infected parts. This response results in disease
resistance. It starts with an oxidative burst and is
thought not only to induce cell death in the host
plant but also to act as signal and to attack the
pathogen. In contrast, in plants that do not
express this type of disease resistance, the patho-
gen is able to establish an infection (Greenberg
and Yao 2004). Such a susceptible interaction also
can depend on host cell death. Especially in inter-
actions between plants and necrotrophic fungi the
induction of programmed cell death in the host is
clearly promoting pathogen growth (reviewed by
Greenberg and Yao 2004).
ROS are not the only substances which can be
released by plants and are directed against the
pathogen (Table 4.1). One example is a-tomatine,
a glycoside produced by tomatoes after infection
with the pathogen Fusarium oxysporum. a-Toma-
tine efficiently induces cell death in the fungus.
The process is accompanied by DNA fragmenta-
tion, depolarisation of the mitochondrial trans-
membrane potential and ROS accumulation.
Treatment with the F0F1-ATPase inhibitor oligo-
mycin, the protein synthesis inhibitor cyclohexi-
mide, or the caspase inhibitor D-VAD-fmk blocks
apoptosis. This kind of cell death is thus not only
dependent on de novo protein synthesis but also
includes mitochondrial-dependent signalling and
energy metabolism (Ito et al. 2007). Based on
these data, it can be assumed that, in such a type
of interaction, an increase in resistance of the
fungus to undergo apoptosis improves the patho-
gen’s capability to infect the host. In fact, this was
clearly demonstrated by heterologous expression
of the gene encoding the anti-apoptotic BCL-2 pro-
tein in the plant pathogen Colletotrichum gloeos-
porioides. Transgenic strains were hypervirulent
and displayed an increased resistance against stres-
ses induced by temperature, ultraviolet (UV) light
and hydrogen peroxide (Barhoom and Sharon
2007). Vice versa, heterologous expression of the
pro-apoptotic BAX protein induced apoptotic cell
death in this fungus. Moreover, the few viable spores
were only able to germinate in a non-pathogenic
way with two germ tubes, even under conditions
that should induce pathogenic germination (one
germ tube) (Barhoom and Sharon 2007).
Apart from a role in host–pathogen interac-
tions, PCD is also induced in antagonistic inter-
actions, interactions in which fungi compete for
nutrients with other species growing on the same
substrate. Secretion of substances inducing apo-
ptotic processes in a competitor provides a major
selective advantage. A variety of substances of this
type have been described and studied in some detail
(Table 4.1). One example is the antifungal protein
PAF produced by Penicillium chrysogenum. PAF
efficiently induces an apoptotic phenotype in
A. nidulans accompanied by hyperpolarisation of
the plasma membrane, PS externalisation and the
accumulation of DNA strand breaks (Leiter et al.
2005). Recently, it was postulated that selective ion
permeability of the plasma membrane is relevant
for the observed morphological changes in suscep-
tible PAF-treated fungi (Marx et al. 2008). PAF and
other similar secreted peptides produced by Asper-
gillus spp. (Gun et al. 1999; Theis et al. 2003) are not
directed against a specific pathogen but efficiently
inhibit growth of a large number of different fungi.
Since neither PAF nor AFP, an antifungal peptide
produced by Aspergillus giganteus, show detrimen-
tal effects on mammalian cells (Szappanos et al.
2005, 2006), these antifungal proteins are promising
candidates for the development of therapies against
human pathogenic fungi (reviewed by Marx et al.
2008). Moreover, PAF in concert with other anti-
fungal compounds is able to inhibit growth of sev-
eral members of the class zygomycetes, members
of which are important post-harvest pathogens of
agricultural products (Galgoczy et al. 2007).
 Apoptosis in Fungal Development and Ageing
Another well analysed apoptosis-inducing
agent is farnesol, a molecule produced by Candida
albicans. Farnesol accumulates at high cell density
and mediates quorum sensing, thereby preventing
C. albicans from switching from yeast to hyphal
growth (Hornby et al. 2001). In a recent study,
farnesol also provided a selective advantage in
antagonistic interactions (Semighini et al. 2006a).
Co-cultivation of C. albicans with A. nidulans dis-
played farnesol-dependent growth impairments
and characteristic features of apoptosis, including
nuclear condensation, accumulation of DNA
strand breaks, PS externalisation and increased
ROS production. Interestingly, the induction of
apoptosis in A. nidulans by farnesol was depen-
dent on poly(ADP-ribose)polymerase (PARP;
Semighini et al. 2006b), an enzyme that in higher
organisms is involved in DNA damage recogni-
tion, DNA repair and induction of apoptosis.
Recently it has been demonstrated that the farne-
sol-dependent induction of apoptosis is not
restricted to A. nidulans but is also observed in
the phytopathogen Fusarium graminearum.
Treatment of growing hyphae with 100–300 mM
farnesol leads to nuclear condensation, DNA
strand breaks and elevated ROS levels (Semighini
et al. 2008).
One interesting type of antagonistic interac-
tion of fungi, the so-called killer phenotype, is
observed in several yeast species, e.g. in Saccharo-
myces cerevisiae, Kluyveromyces lactis (reviewed
by Schmitt and Breinig 2006), in members of the
genus Pichia and in Wingea robertsiae (Klassen
and Meinhardt 2003; Klassen et al. 2004). The
induction of cell death by these toxins appears to
be linked to the execution of apoptotic processes.
Depending on the genotype, some natural strains
Table 4.2. Influence of apoptosis factors on fungal development
Species Apoptosis factor
A. nidulans Over-expression of Parp
C. gloeosporioides Expression of human anti-
apoptotic Bcl-2
C. cinereus Four different types of mutations Impairment of spore formation,
P. anserina Deletion of both metacaspase
genes
S. cerevisiae pheromone treatment in the
absence of mating partner
69
of these yeasts produce a killer toxin that leads to
the death of susceptible strains. The toxin-pro-
ducing strains are resistant against their own
toxin (reviewed by Schmitt and Breinig 2006). In
a 2005 study, moderate doses of the S. cerevisiae
toxins K1 and K28 and the Zygosaccharomyces
bailii toxin zygocin trigger ROS production and
apoptosis mediated via YCA1, the single metacas-
pase known in yeast, in susceptible S. cerevisiae
strains (Reiter et al. 2005). The same holds true for
the pPac1-2 toxin of P. acaciae. Treatment of
S. cerevisiae with this toxin results in cell death
accompanied by DNA strand breaks, PS externa-
lisation and the formation of ROS (Klassen and
Meinhardt 2005).
Not only fungi and other eukaryotes but also
prokaryotes appear to be able to induce apoptosis
in fungi. From investigations in which yeast cul-
tures have been treated with acetic acid and found
to undergo apoptosis, it has been speculated that
acetic acid secreting bacteria have an advantage in
nature when competing with yeast for available
nutrients (Ludovico et al. 2002).
B. Apoptosis During Fungal Reproduction
Beside the well established role of apoptosis in
fungal inter- and intraspecies interaction, only a
few observations indicate a role of this suicide
programme in fungal reproduction (Table 4.2).
In yeast, the first step in sexual reproduction is
the recognition of a pheromone secreted by a
partner of one mating-type by a receptor on the
surface of a partner of the opposite mating-type.
Impairments of the subsequent step, fusion of
two cells of the opposite mating-type, induce
Phenotype References
Impairment of conidiophore development Semighini et al.
(2006b)
Increased conidiospore production Barhoom and
Sharon
(2007)
Lu (2000), Lu
basidia-specific apoptosis et al. (2003)
Impairment of fruiting body development Hamann et al.
(2007)
ROS production, DNA breakage, Severin and
mitochondria-dependent apoptotic Hyman
cell death (2002)
70 Diana Brust et al.
mitochondria-dependent apoptotic cell death
accompanied by ROS production and DNA break-
age (Severin and Hyman 2002). Yeast cells of mat-
ing-type a were treated with the a pheromone of
the opposite mating-type. After induction of the
sexual reproduction pathway, the physical ab-
sence of the appropriate mating partner induced
ROS production suggesting that failure of mating
induces apoptosis. Death of cells has been sug-
gested to represent an altruistic event eliminating
in nature those cells that are unable to mate
(Severin and Hyman 2002). Interestingly, both
the formation of diploid yeast cells and the induc-
tion of apoptosis in cells induced by the a phero-
mone but not able to fuse with a mating partner
are dependent on kinase STE20 function (Schrick
et al. 1997; Severin and Hyman 2002). A mecha-
nistic link of these two pathways, sexual reproduc-
tion and apoptosis, seems to be related to large-
scale chromatin remodelling. Fragmentation of
nuclear DNA during apoptosis is strictly related
to phosphorylation of serine 10 of histone H2B, a
process catalysed by STE20 (Ahn et al. 2005a).
Interestingly, the same histone modification is
observed during meiosis, the next step during
sexual reproduction after the formation of
diploids (Ahn et al. 2005b). H2B mutants mimick-
ing a constitutive phosphorylation of Ser10 show a
drastic reduction of growth. Surviving cells exhibit
a cellular morphology similar to hydrogen peroxide-
treated wild-type cells (Ahn et al. 2005a). Chroma-
tin remodelling plasticity is speculated to be
involved in cell survival (Ahn et al. 2005a). During
apoptosis chromatin becomes condensed. This
condensation ultimately leads to cell death. Signif-
icantly, cytological analyses revealed that chroma-
tin condensation during meiosis is not a static
event. Especially in prophase I of meiosis, chro-
matin globally cycles between expanded and con-
tracted stages (reviewed by Kleckner et al. 2004).
It thus appears that this type of cycling inhibits
the induction of apoptosis. However, a more re-
cent study questions the role of apoptosis in pher-
omone-induced cell death. In this study, the effect
of a range of pheromone concentrations on yeast
cells was investigated (Zhang et al. 2006). High
pheromone concentrations induced rapid cell
death, requiring the presence of the transmembrane
protein FIG1, which promotes Ca2+-independent
cell death. Lower concentrations of pheromone
induced a second wave of cell death and were
dependent on cell wall degradation. Significantly,
several hallmarks of apoptosis were not observed
in cells undergoing death (Zhang et al. 2006).
Additional data suggest an impact of apopto-
sis on development of fungi other than yeast. In
the basidiomycete Coprinopsis cinereus four dif-
ferent types of mutants impaired in meiosis or
spore formation were isolated and analysed. In
group one, meiosis can be completed; however,
the formation of spores is affected. In this group,
apoptotic processes are induced at the tetrad stage
of basidiospore formation. The three other groups
of mutants have in common a defect in prophase
of meiosis. In all groups typical markers of apoptosis
were observed, including chromatin condensa-
tion, DNA fragmentation, cytoplasmic shrinking
and DNA degradation (Lu et al. 2003). In another
fungus, the filamentous ascomycete Podospora
anserina, circumstantial evidence for a role of
apoptosis factors in meiospore development was
obtained in a mutant in which two metacaspase
genes were deleted. This mutant is severely im-
paired in the formation of ascospores and fruiting
bodies (Hamann et al. 2007).
Finally, some recent data also suggest a role of
apoptotic processes in asexual reproduction.
Using fungal extracts of conidiospore producing
cultures of A. nidulans, in vitro degradation of
PARP was demonstrated (Thrane et al. 2004). In
these cultures, the amount of PARP is increased
(Thrane et al. 2004) due to a strong transcriptional
upregulation (Semighini et al. 2006b). A regulated
expression of this gene appears to be essential for
conidiospore development because overexpres-
sion of Parp leads to impairments of conidiophore
development. As a consequence, a 104-fold re-
duction in spore numbers was found in the
corresponding mutants (Semighini et al. 2006b).
Currently it is not clear whether the influence of
PARP is due to apoptotic processes or is the result
of an indirect role of this enzyme. However, a
direct involvement of apoptosis in mitospore for-
mation is supported by experiments with the plant
pathogen C. gloeosporioides: overexpression of the
gene encoding the human anti-apoptotic BCL-
2 protein not only led to hypervirulence but also to
an enhanced conidiospore production (Barhoom
and Sharon 2007).
Beside the role in reproduction, apoptosis and
autophagy also play an important role in fungal
non-self-recognition between different isolates of
the same species. After fusion of two genetically
incompatible hyphae, a cell death reaction, termed
 Apoptosis in Fungal Development and Ageing 71

‘heterokaryon incompatibility’, is induced (reviewed
by Glass and Dementhon 2006). In Neurospora
crassa this reaction displays clear characteristics of
apoptosis (Marek et al. 2003). In P. anserina, auto-
phagy is strongly induced during the incompatibility
reaction, antagonizing pro-death signals (Pinan-
Lucarré et al. 2003; Pinan-Lucarré et al. 2005;
reviewed by Pinan-Lucarré et al. 2007). Interestingly,
P. anserina autophagy mutants are impaired in
female fertility, linking this process to sexual devel-
opment (reviewed by Pinan-Lucarré et al. 2007).
C. The Role of Apoptosis in Fungal
Lifespan Control
While it is clear that apoptosis plays an important
role in differentiation and development, much less
is known about its specific role in ageing and life-
span control of biological systems. Only in the past
decade have some clues for such a role emerged,
some of which have been derived from investiga-
tions of fungi as experimentally tractable ageing
models. In general, these studies revealed a connec-
tion between age-related symptoms and apoptosis
induction (Table 4.3). Prevention of apoptosis has
a clear lifespan-prolonging effect. One example for
this connection is the increased viability of yeast
cells in stationary phase that express the human
Bcl-2 gene (Longo et al. 1997). Another is that
chronologically aged yeast cultures display charac-
teristic markers of apoptosis, including DNA frag-
mentation, PS externalisation and chromatin
condensation, markers which are not observed in
exponentially growing cultures (Fabrizio et al.
2004; Herker et al. 2004). The involvement of apo-
ptosis in chronological ageing is further stressed by
the observation that overexpression of Yap1, one
key regulator of tolerance against oxidative stress,
leads to increased survival of the transgenic strains
in stationary culture (Herker et al. 2004). The same
holds true when the gene encoding yeast metacas-
pase YCA1 is deleted (Herker et al. 2004). More-
over, prolonged cultivation of Yca1 overexpressing
strains results in a pronounced reduction in viabil-
ity after 44 h or 96 h of cultivation, accompanied by
the appearance of different apoptotic markers. This
effect is not observed when a mutant form of Yca1
containing a serine residue instead of a cysteine in
the catalytic centre of the protein is overexpressed
(Madeo et al. 2002). During the past five years,
additional findings have accumulated demonstrat-
ing that different apoptotic pathways are involved
in the control of chronological ageing in yeasts
(Wissing et al. 2004; Li et al. 2006; Barhoom and
Sharon 2007; Büttner et al. 2007).
Interestingly, markers of apoptosis including
PS externalisation and DNA strand breaks occur

Table 4.3. Factors implicated in apoptosis and fungal lifespan control

Species Factor Influence on lifespan References

C. gloeosporioides Expression of human anti-apoptotic Increase in chronological
Bcl-2 lifespan
P. anserina Deletion of metacaspases Increase in replicative lifespan
Deletion of fission factor PaDnm1 Increase in replicative lifespan
S. cerevisiae Expression of human anti-apoptotic Increase in chronological
Bcl-2 lifespan
Over-expression of Yca1 Increase in chronological
lifespan
Deletion of Mmi1 Increase in replicative lifespan
Deletion of fission factors Dnm1 and Increase in replicative lifespan
Fis1
Over-expression of EndoG Decrease in chronological
lifespan
Deletion of Aif1 Increase in chronological
lifespan
Disruption of Ndi1 Increase in chronological
lifespan
Deletion of Kex1 Increase in chronological
lifespan
Deletion of Isc1 Decrease in chronological
lifespan
Barhoom and Sharon
(2007)
Hamann et al. (2007)
Scheckhuber et al. (2007)
Longo et al. (1997)
Madeo et al. (2002)
Rinnerthaler et al. (2006)
Scheckhuber et al. (2007)
Büttner et al. (2007)
Wissing et al. (2004)
Li et al. (2006)
Hauptmann and Lehle
(2008)
Almeida et al. (2008)
72 Diana Brust et al.
also during replicative ageing of yeast cells (Laun
et al. 2001). Moreover, an increase of yeast repli-
cative lifespan by deletion of the yeast gene coding
for the homologue of TCTP (a human protein
interacting with BCL-XL) was reported,
(Rinnerthaler et al. 2006). The details of the un-
derlying mechanism are poorly understood.
Recently, data on the impact of apoptosis on
ageing were reported in another fungal ageing
model, P. anserina. Since this process occurs dur-
ing active growth of cultures and depends on
replication of the genetic material, it also repre-
sents a type of replicative ageing. In P. anserina,
ageing can easily be followed macroscopically by
analysing growth of cultures on solid medium.
Depending on the genetic background and culture
conditions, the mean lifespan of a wild-type cul-
ture is two to four weeks. Various molecular path-
ways are involved in lifespan control in this
fungus. Among others these are: the type of respi-
ration (Dufour et al. 2000; Borghouts et al. 2001;
Lorin et al. 2001; Krause et al. 2004; Stumpferl
et al. 2004; Sellem et al. 2005; Krause et al. 2006;
Sellem et al. 2007), mitochondrial DNA stability
(Stahl et al. 1978; Cummings et al. 1979; Belcour
et al. 1981; Kück et al. 1981; Osiewacz and Esser
1984; Kück et al. 1985), dietary restriction (Tud-
zynski and Esser 1979; Maas et al. 2004; Maas et al.
2007), copper metabolism (Borghouts et al. 1997;
Borghouts and Osiewacz 1998; Borghouts et al.
2000; Stumpferl et al. 2004), translation fidelity
(Belcour et al. 1991; Silar and Picard 1994), mito-
chondrial protein import (Jamet-Vierny et al.
1997), mitochondrial dynamics (Jamet-Vierny
et al. 1997; Scheckhuber et al. 2007). Interestingly,
recent investigations identified apoptotic machin-
ery in P. anserina which also was found to be
involved in lifespan control. Deletion of the
genes encoding the two metacaspases, PaMca1
and PaMca2, resulted in an increase in mean life-
span, suggesting that apoptosis is also induced in
the final stage of senescence in this fungus. Most
pronounced is the effect of the PaMca1 deletion,
leading to a 2.5-fold lifespan increase. Important-
ly, protoplasts of 15-day-old cultures of the
PaMca1 deletion strain display a higher viability
after treatment with hydrogen peroxide than pro-
toplasts of the wild-type strain (Hamann et al.
2007). Moreover, PaMca1 deletion leads to a
pronounced resistance against the apoptosis-in-
ducing agent etoposide (Scheckhuber et al. 2007),
suggesting that metacaspases are involved in
apopototic cell death and fungal senescence. At
least PaMCA1, one of the two metacaspases of P.
anserina, becomes activated during senescence
leading to apoptotic cell death. High levels of
hydrogen peroxide can be detected in senescent
mycelium and because this kind of ROS induces
PS externalisation in P. anserina, PaMCA1 seems
to be activated by ROS. PaMca1 deletion strains
are not characterised by reduced ROS levels, but
rather by impairment to respond to ROS and to
induce apoptosis (Hamann et al. 2007). This idea
is supported not only by the lifespan extension of
the deletion strains, but also by the demonstration
of a metacaspase-dependent peptidase activity in
senescent mycelium (Hamann et al. 2007).
One important issue that remains to be
solved in order to better understand the impact
of apoptosis on ageing and lifespan control is to
unravel the intracellular signalling pathways
involved in the induction of apoptosis. In yeast,
the dynamics of the actin cytoskeleton plays an
important role. First, a decrease in actin turn-
over, which can be experimentally induced by
specific mutations in actin or treatment with
actin-stabilising drugs, was observed in aged
cells (Gourlay et al. 2004). Second, an increase
in actin turnover via deletion of the Scp1 gene
encoding the actin bundling protein SCP1 leads
to lifespan extension of dividing and non-divid-
ing yeast cells (Gourlay et al. 2004). The role of
actin dynamics is linked to the Ras-cAMP signal-
ling, a pathway which is an important regulator
of longevity. Constitutive Ras-cAMP activation
impairs scavenging of mitochondrial deficiency
and oxidative stress and reduces lifespan (Hee-
ren et al. 2004). A link between Ras-cAMP signal-
ling and actin is provided by the phenotype of
yeast mutants lacking either of the two actin
regulatory proteins SLA1 or END3. The charac-
teristics of these strains resemble those of strains
in which Ras-cAMP signalling is constitutive.
The corresponding mutants are characterised
by elevated ROS production, mitochondrial dys-
function and reduced longevity. Interestingly,
ROS levels and lifespan of these strains can be
reverted to wild-type characteristics via overex-
pression of Pde1, a negative regulator of the Ras-
cAMP pathway (Gourlay and Ayscough 2005).
Recently, a direct link between oxidative
stress and the actin cytoskeleton was obtained
by analysis of the yeast old yellow enzymes
OYE2 and OYE3. OYE2 was demonstrated to
 Apoptosis in Fungal Development and Ageing
interact with actin (Haarer and Amberg 2004).
An almost complete knockdown of Oye2 resulted
in defects in cytoskeletal organization (Haarer
and Amberg 2004). Moreover, simultaneous dele-
tion of Oye2 and the gene encoding glutathione
oxidoreductase GLR1 increases strongly the mor-
phological defects (Odat et al. 2007), suggesting
that OYE2 protects actin from oxidative damage.
Based on these findings OYE proteins are consid-
ered to be important members of a signalling
network connecting ROS generation, actin cyto-
skeleton dynamics and cell death.
In addition to actin cytoskeleton dynamics,
microtubule dynamics seem to play a role in
the induction of apoptosis. Deletion of Mmi1,
the gene encoding the yeast homologue of the
human BCL-XL binding protein TCTP, leads to
increased resistance against hydrogen peroxide
and a small, but significant increase in replicative
lifespan (Rinnerthaler et al. 2006). This pheno-
type is accompanied by an increased sensitivity
against the microtubule-destabilising agent ben-
omyl suggesting that MMI1 interacts with micro-
tubules.
Finally, another important contributor to a
‘healthy’ cellular status, dynamics of mitochon-
dria, appears to be linked to apoptosis. This
conclusion is derived from recent investigations
in which the mitochondrial fusion and fission
machinery was manipulated in both S. cerevisiae
and P. anserina (Fannjiang et al. 2004; Scheck-
huber et al. 2007). Deletion of genes encoding
mitochondrial fission factors (DNM1 and FIS1
in yeast; DNM1 in P. anserina) retard ageing
and increase ‘health span’, the healthy period
of time within the life cycle of organisms. Dur-
ing the ageing of wild-type strains, mitochon-
dria change their structure from networks to
punctuate entities. However, this change occurs
only in the very last period of the lifetime of this
fungus. Although in yeast the deletion of fission
factors has an effect on mean lifespan less pro-
nounced than in P. anserina, yeast mutants
maintain a generation time comparable to that
of young cells, even after 30–40 generations, an
indicator of a ‘healthy’ cell status. A clear link
between mitochondrial dynamics and apoptotic
processes is provided by an increased resistance
of the PaDnm1 deletion strain of P. anserina
against etoposide, an elicitor of apoptosis inde-
pendent of pro-apoptotic Ca2+ waves (Scheck-
huber et al. 2007).
73
III. Concluding Remarks and
Future Directions
Within the past decade, apoptosis in fungi emerged
as a molecular mechanism that is linked to differ-
ent biological processes. Of special applied interest
is the impact of apoptotic processes of pathogenic
fungi with their hosts because these kinds of inter-
actions represent a major threat for human health
as well as for food production. For example, in a
recent study, the incidence of blood stream infec-
tions with species of the Candida genus was deter-
mined in a population-based active surveillance
program of two areas in the United States (Hajjeh
et al. 2004). Compared to earlier studies, the overall
incidence of candidemia was higher. Another ex-
ample is rice blast caused by the plant pathogen
Magnaporthe grisea, which is responsible for 25%
annual yield loss of rice production in Japan (Ribot
et al. 2008). Research to understand the underlying
basis of host-pathogen interactions (including
the details involved in the induction of apoptotic
processes) are of great relevance towards the devel-
opment of efficient fungicides and effective anti-
fungal therapies.
Apoptosis in fungi and mammals clearly share
characteristic features; however, it is now also
obvious that the underlying mechanisms differ in
a number of details. As one example, fungi lack
biochemical homologues of caspases, aspartate-
specific cysteine proteases which are among the
main players in mammalian apoptosis. Instead,
they possess metacaspases, cysteine proteases
which seem to originate from the same ancestral
gene as caspases (Uren et al. 2000) and are proba-
bly functional homologues of caspases. Although
classic caspase substrates are cleaved by fungal
extracts (Madeo et al. 2002; Mousavi and Robson
2003; Thrane et al. 2004), it is unlikely that these
substrates are metacaspase substrates. Plant, pro-
tozoan and fungal metacaspases display Arg-Lys
protease specificity instead of the Asp specificity
characteristic of caspases (Vercammen et al. 2004;
Watanabe and Lam 2005; Gonzalez et al. 2007;
Hamann et al. 2007). Obviously, additional work
is required to elucidate the role of these proteases
in fungal apoptosis in more detail. One important
piece of information, the identity of the cellular
substrate of metacaspases, could be obtained from
a proteome analysis of wild-type and metacaspase-
deletion strains. Because of the clear relationship
between metacaspases and caspases, the study of
74 Diana Brust et al.
fungal metacaspases could provide a key platform
for understanding how apoptotic mechanisms
evolved.
As described in this chapter, apoptotic path-
ways are linked to some other molecular and cel-
lular pathways. However, the currently available
data about these links are rather fragmented and
descriptive. In particular, among other aspects, it
will be important to elucidate the mechanistic
details linking the role of the cytoskeleton, mito-
chondrial dynamics, ROS generating and/or scav-
enging pathways with apoptotic processes. In
general, a more detailed view incorporating more
of the individual components of the involved
pathways will help to better understand apoptosis
as a ‘molecular module’ of complex biological
processes such as the interaction of fungi with
other organisms, fungal development or ageing
and lifespan control. Finally, investigations
aimed at testing the general applicability of a
newly elucidated pathway and its conservation
seem to be relevant. In such approaches, micro-
organisms like fungi are experimentally tractable
systems that can serve as model systems to deci-
pher basic mechanisms, which – due to practical
or ethical reasons – cannot be approached directly
in higher systems.
Acknowledgements. The authors gratefully acknowledge
the European Commission (LSHM-CT-2004-512020) and
the Deutsche Forschungsgemeinschaft (Bonn, Germany)
for supporting experimental work referenced from the
authors’ laboratory.
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5 Communication of Fungi on Individual, Species, Kingdom,
and Above Kingdom Levels

URSULA KÜES1, MÓNICA NAVARRO-GONZÁLEZ1

CONTENTS messages are transmitted from a sender to a

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 receiver by means of a suitable medium. Commu-
II. Communication Within and Between nication is an obvious instrument used to co-
Fungal Colonies – Vegetative Interactions . . . . . 80 ordinate human and animal societies.
A. Communication in Mediating
Vegetative Fusions Within Communication is highest developed in the animal king-
Fungal Colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 dom where it involves – to variable degrees – the five
B. Communication in Mediating senses sight, hearing, touch, smell, and taste.
Vegetative Fusions Between Different
Individuals of Filamentous In a broader view, communication is not limited to
Ascomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 the animal kingdom. Every interchange and ex-
C. Communication in Mediating
Vegetative Fusions Between Different
change of information between living cells can be
Individuals of Basidiomycetes . . . . . . . . . . . . . 81 considered to be communication. Accordingly,
D. Communication in Population Growth there is communication in all types of living organ-
Control and Germination . . . . . . . . . . . . . . . . . . 84 isms, from the bacteria up to the highest developed
E. Communication in Dimorphism and eukaryotes. In nature, communication occurs man-
Asexual Reproduction . . . . . . . . . . . . . . . . . . . . . . 86
III. Communication Between Fungi in
ifold on different cellular levels: between cells of a
Sexual Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 same individual, between individuals of a species,
A. Communication in Mating . . . . . . . . . . . . . . . . . 87 and between organisms of different species within
B. Communication in Fruiting Body and above taxonomical clades up to interkingdom
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 communication. Communication has many func-
IV. Communication Between
Fungi and Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
tions and serves e.g. in nutrient absorption and
V. Communication Between disposition, in reproduction and recombination,
Fungi and Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 in distribution of species, and in the co-ordinated
VI. Communication Between formation of ecological communities including
Fungi and Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 also the targeted suppression of competitors.
VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Concerning bacteria, lower eukaryotes in-
cluding the fungi, and plants, modes of intra-
and interspecies communication appear to be
mainly of chemical nature (Camilli and Bassler
2006; Dudareva et al. 2006; Bouwmeester et al.
I. Introduction 2007; Thakeow et al. 2007; Lowery et al. 2008;
Ryan and Dow 2008; this chapter). Electrophysi-
In general terms, communication is understood as cal mechanisms exist also but are little studied. A
a process by which information is exchanged new research area “plant neurobiology” focuses
between living individuals through a common on intercellular long-distance communication in
system of symbols, signs, tones or behaviour, i.e. plants based on bioelectrochemical signals,
which is seen as an analogous development to
1
the neurosystems in the animal kingdom
Division of Molecular Wood Biotechnology and Technical My-
cology, Büsgen-Institute, Georg-August-University Göttingen,
(Volkov 2000; Brenner et al. 2006; Barlow 2008).
Büsgenweg 2, 37077 Göttingen, Germany; e-mail: ukuees@gwdg. Electrical fields appear to be also used in
de, mnavarr@gwdg.de communication between plants and oomycete

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
80 Ursula Kües and Mónica Navarro-González
Fig. 5.1. Hyphal fusion (anastomosis) in a culture of Coprinopsis stercorarius strain CBS 477.70 occurred between short
hyphal side branches (see open arrows) whilst other side branches grow with their tips towards each other (filled arrow)
plant pathogens, respectively between plants and
arbuscular mycorrhizal (AM) fungi (Berbara
et al. 1995; van West et al. 2002; Ramos et al.
2008). Bioelectric currents may also serve in in-
tercellular communication in fungi, particularly
in filamentous species (Harold et al. 1985, Gow
1989, Potapova 2004), but deeper biophysical
research into this in fungi is necessary.
Research in fungi detected in recent years
various types of extracellular signalling molecules
(“infochemicals”) that act or may act at a distance
in biological signalling between fungal cells within
a fungal individual, between fungal individuals of
the same or other species and between fungi and
other groups of organisms.
Biological aspects of signalling within and between fungal
colonies were recently reviewed in a number of excellent
chapters in the second edition of The Mycota, Vol. 1
(Casselton and Challen 2006; Glass and Fleissner 2006;
Feldbrügge et al. 2006; Pöggeler et al. 2006; Schimek and
Wöstemeyer 2006; Ugalde 2006). For further reading on
biological aspects on communication of fungi and other
groups of organisms, the reader is referred to the topical
reviews by Chiron and Michelot (2005), Bouwmeester et al.
(2007), and Thakeow et al. (2007), respectively.
II. Communication Within and Between
Fungal Colonies – Vegetative
Interactions
A. Communication in Mediating Vegetative
Fusions Within Fungal Colonies
Buller (1933) recognized already in the early last
century that within mycelial fungal colonies there
can be a functional division of hyphae – some of
them act as leading hyphae in colony growth for
occupying new substrates, others are formed by dif-
ferent order branching from the leading hyphae and
their main branches lead to a network of hyphae in
which communication and transport ways are dra-
matically shortened. To form a well interconnected
network that eases nutritional translocation within
the mycelium (Rayner 1991, Rayner et al. 1994,
Olsson and Gray 1998), hyphae of higher branching
order appear to grow within a colony towards each
other in order to fuse by a process known as anasto-
mosis (Fig. 5.1). The targeted growth towards each
other – variously called positive autotropism, hyphal
homing, or remote sensing - starts over certain
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels
distances, suggesting that the fungi are aware of
other cells of the colony growing in their neighbour-
hood (Gregory 1984; Hickey et al. 2002; Glass et al.
2004; Glass and Fleissner 2006). Most likely, intercel-
lular chemical signalling is taking place between
different hyphae of a colony but the nature of such
signalling molecules has not yet been discovered in
any fungus (Ugalde 2006).
Bhuiyan and Arai (1993) reported as an indirect evidence
for such communication that elimination of water soluble
substances from the environment blocks hyphal attraction
and fusion in the basidiomycete species Rhizoctonia oryzae.
B. Communication in Mediating Vegetative
Fusions Between Different Individuals
of Filamentous Ascomycetes
Substances acting in hyphal attraction within a
colony may be the same than those functioning
in homing between hyphae of genetically alike
colonies or between hyphae and (germinated)
spores of the same genotype (Glass et al. 2004).
In the ascomycete Neurospora crassa, attraction
between genetically identical spore germlings was
studied by Read and colleagues, using microma-
nipulation by optical tweezers. With this tech-
nique, through application of laser beams, the
position of a spore or a germling can be altered
relative to another spore or spore germling
(Fleissner et al. 2005; Roca et al. 2005a; Wright
et al. 2007). In such way, homing of conidial
anastomosis tubes (CATs) has unambiguously
been documented in N. crassa (Roca et al. 2005a).
CATs are thin, short, and unbranched hyphae of determined
growth that are only formed on ascomycete conidia when
spores are present in high densities. CATs home towards
each other and fuse, which is different from the larger germ
tubes that avoid each other by growth (Roca et al. 2005b;
Glass and Fleissner 2006).
In N. crassa, the tips of CATs were shown to be
both the sites of chemo-attractant secretion and of
perception. So far, analysis of a specific cAMP-
defective N. crassa mutant (strain cr-1) excluded
cAMP as the attractant (Roca et al. 2005a). Spec-
ulations are presented that the yet unknown
chemo-attractants might be peptides, which is
supported by the pertinent argument that pep-
tides allow a higher specificity. CATs have been
described in over 70 ascomycetous species (Roca
et al. 2005b). CAT homing is however not restricted
81
to one kind of spores but it can happen also be-
tween spores of different species, even up to CAT
fusion (Köhler 1930; Ishitani and Sakaguchi 1956).
At least in N. crassa, CAT homing as well as CAT
fusion can occur between vegetative incompatible
strains, albeit the latter occurs at a reduced level.
Vegetative incompatibility reactions upon CAT
fusions happen much later compared with those
that follow fusion of vegetative hyphae in mature
colonies (Roca et al. 2005a, b).
Vegetative incompatibility, also known as heterokaryon in-
compatibility, refers to a phenomenon where fusion between
vegetative hyphal cells of different genetic background leads
with the resulting heterokaryosis to cellular death (for
further reading, see Glass et al. 2000; Esser 2006).
C. Communication in Mediating Vegetative
Fusions Between Different Individuals
of Basidiomycetes
Mating in the basidiomycetes is governed in the
bipolar species by one mating type locus and in
the tetrapolar species by two mating type loci
(usually called A and B). For successful mating
leading from sterile monokaryons to fertile dikar-
yons, two fusing strains need to be compatible, i.e.
of different mating type (further information
given by Casselton and Challen 2006; Casselton
and Kües 2007; Sect. III.A). In species of higher
basidiomycetes, hyphal fusions occur however
between all kinds of monokaryotic mycelia,
irrespectively of whether being of the same or of
different mating types (Sicari and Ellingboe 1967;
Ahmad and Miles 1970a, b; Leary and Ellingboe
1970; Fig. 5.2) and all attempts to isolate secreted
substances from fungal cultures specifically acting
in mating failed (Hiscock and Kües 1999). Accord-
ingly, it was repeatedly assumed that there is no
extracellular mating-signal reaction before cellu-
lar fusion (e.g. see Kothe 1996, 2008; Vaillancourt
et al. 1997; Casselton and Olesnicky 1998). Fre-
quencies of hyphal fusion in the tetrapolar Schi-
zophyllum commune were reported to be higher
between colonies with different A mating types
than between strains sharing the same A mating
type (Ahmad and Miles 1970a). In contrast, in
Coprinopsis cinerea frequencies of hyphal fusion
were said to be higher in confrontations of strains
with different B mating types than between com-
binations of strains with the same B mating type
(Smythe 1973). Other authors found with respect
82 Ursula Kües and Mónica Navarro-González
Fig. 5.2. Hyphal interactions in confronting mycelia of a
compatible cross of Coprinopsis cinerea strains PS004-1
(mating type A3, B42; hyphae marked at the left or below
with white symbol *) and PS004-2 (mating type A42, B1;
hyphae marked at the left or below with black symbol *).
Fusions can be seen between side branches of the same
monokaryon (black circles) and between side branches of
the compatible strains (white circles). Not all co-incidences
between two hyphae of the same strain (dotted black cir-
cles) or of different monokaryons (dotted white circles)
end in hyphal fusion – hyphae rather overgrow each
other. For observing hyphal events in confrontations of
mating compatible strains, the two monokaryons were
inoculated at about 1 cm distance on a thin film of yeast
extract/malt extract/glucose agar medium solidified on a
to mating types no apparent differences in fre-
quencies of hyphal fusions in C. cinerea (Sicari
and Ellingboe 1967) and there are reports on mu-
tual attraction of hyphae in S. commune irrespec-
tive of mating type (Ahmad and Miles 1970a;
Raudaskoski 1998). Hyphal fusions in S. commune
are stimulated by a secreted factor that acts equally
well on combinations of mating-compatible
strains and on combinations of mating-incompat-
ible strains. The effective factor is however only
induced in confrontations of mating-compatible
strains whilst hyphal fusion is not required for its
production (Ahmad and Miles 1970a). Further-
more, in Lenzites betulinus a secreted substance
appears to cause formation of a “répulsion bar-
rage” (“sexual barrier”, a zone of poor growth
between colonies caused by an aversion for one
another) between colonies having the same B mat-
ing type (Hennebert et al. 1994).
microscope slide. A middle zone of agar about 0.7 mm in
width was eliminated in between the two inocula prior to
incubation at 37
C. After 24 h incubation both strains
grew into the free window towards each other. The whole
area was photographed in small sections (one after the
other) which subsequently were composed into a larger
collage that unambiguously allowed to follow up the origin
of leading hyphae (the thicker hyphae in the photograph)
and their thinner sub-branches and to correctly assign
them to one of the two strains. The photograph shows a
subsection of the collage where hyphae of the two strain
intermingle and have undergone some self- or non-self-
fusion events. Note at the right side the oidiophore
(marked by an “o”) that typically form in aerial mycelium
monokaryons of C. cinerea (Polak et al. 2001)
Different categories of hyphal fusions are observed between
confronting colonies: tip-to-tip (between two hyphal tips),
tip-to-side (between a hyphal tip and a lateral hyphal wall),
tip-to-peg (between a hyphal tip and a lateral swelling of a
hypha), and peg-to-peg fusions (between lateral swellings of
two neighbouring hyphae; Buller 1933; Smythe 1973).
According to Raudaskoski (1998), tip-to-tip and tip-to-
side fusions occur frequently in confrontations of S. com-
mune strains independently of mating types and in these
fusions one of the hyphae appears to be attracted by the
partner hypha. At later stages, particularly in confrontation
of strains with different B mating types, peg-to-peg fusions
occur in higher frequencies. The two pegs at neighbouring
hyphae appear to stimulate each other in growth. Raudas-
koski (1998) suggested that such high frequency of peg-to-
peg fusions may come from the action of mating type
pheromones encoded in the B mating type locus. Thus,
Raudaskoski (1998) distinguished potential vegetative hy-
phal interactions from potential sexual hyphal interactions.
As described in S. commune (Voorhees and
Peterson 1986; Todd and Aylmore 1985), Polyporus
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels
dryophilus (Morton and French 1970), and Laccaria
laccata (Fries 1983a), attraction is also possible
between basidiospores and dikaryotic hyphae or
monokaryotic and dikaryotic hyphae. In C. cinerea
and other Coprini, uninucleate mitotic spores
(oidia) are known to generally attract monokaryotic
hyphae of the same and sometimes also of related
species (Kemp 1975, 1977). Homing reactions
within and between closely related species were
described by Fries (1981, 1983b) between basidio-
spores and dikaryotic hyphae in the genus Lecci-
num. Interspecies homing by mitotic and meiotic
spores results regularly in somatic incompatibility
reactions, leading to the death of the foreign
hyphae (Kemp 1977; Fries 1981), whereas in intra-
species reactions nuclei of one of the fusing part-
ners might be degraded (Todd and Aylmore 1985).
Particularly the observations of homing reactions
argue for extracellular communication between
different individuals of higher basidiomycetes.
However, the controversial results and various
types of observations do not allow us to deduce a
clear-cut picture on the situation whether there is
only vegetative communication prior to hyphal
fusion or whether there is in addition a sexually
determined communication (Raudaskoski 1998;
Hiscock and Kües 1999). Vegetative and sexual
reactions aiming at different targets (e.g. ease of
translocation, supply of nutrients vs sexual repro-
duction) might just be overlapping and this would
experimentally not be easy to separate (Hiscock
and Kües 1999; Kües 2000). Figure 5.2 shows
hyphae in the confrontation zone of two compati-
ble monokaryons of C. cinerea. We found in close
vicinity fusions between hyphae of the same and
between hyphae of different mating type. The first
case of fusions can be considered vegetative but
whether the latter is vegetative or sexual cannot be
distinguished. Hyphae may overgrow others with-
out fusing to them regardless of whether they share
the mating type or not (Fig. 5.2). The observations
imply that not all hyphae are competent for fusion
or that growing hyphae do not recognise all poten-
tial self or non-self partners due to a lack of com-
munication between the hyphae.
Sexual development in higher basidiomycetes
in part is regulated by the B mating type genes
encoding small peptide pheromones and phero-
mone receptors of the seven-transmembrane G pro-
tein-coupled type (Casselton and Challen 2006;
Casselton and Kües 2007). Mating-type specific
peptide pheromones occur also in ciliates such as
83
Euplotes raikovi. The E. raikovi pheromones have
both paracrine (non-self) functions by binding to
mating-type-specific pheromone receptors of cells
of different mating type as well as autocrine (self)
functions by binding to pheromone receptors of the
cell they are secreted from (Luporini et al. 2005,
2006). This finding provoked the idea that autocrine
and paracrine reactions by pheromones might hap-
pen also in the higher basidiomycetes (Hiscock and
Kües 1999; Kües 2000). A paradigm of autocrine
and paracine reactions of mating-type pheromones
has indeed subsequently been found in the basidio-
mycetous yeast Cryptococcus neoformans (Shen
et al. 2002; Lin et al. 2005).
Binding of mating-type pheromones from
higher basidiomycetes to pheromone receptors
of another mating-type has been demonstrated
by heterologous expression in baker’s yeast (Fowler
et al. 1999; Hegner et al. 1999; Olesnicky et al. 1999).
It might be possible that in the native hosts, the
mating-type specific pheromone-receptors bind
the pheromones of the own mating type less strin-
gent than competitive pheromones of different mat-
ing type, in a manner as described for autocrine and
paracrine pheromone binding in the ciliate E. rai-
kovi (Luporini et al. 2005, 2006). However, there are
increasing reports on occurrence of genes in the
basidiomycetes for potential pheromone receptors
that are not mating-type-linked (James et al. 2004,
2006; Aimi et al. 2005; Niculita-Hirzel et al. 2008;
Martinez et al. 2009). Moreover, in the genomes of C.
cinerea and Laccaria bicolor, large families of genes
for closely related, non-mating type, pheromone-
like peptides were recently discovered (Niculita-
Hirzel et al. 2008; Fig. 5.3). Both, the non-mating-
type pheromone receptors and the pheromone-like
peptides could be candidates for functioning in veg-
etative communication within a species and also
between species. Experimental data will have to
prove that.
Different from the highly dissimilar mating-type phero-
mones, the non-mating-type pheromone-like peptides of
C. cinerea and L. bicolor are comparably well conserved in
sequence (Niculita-Hirzel et al. 2008; Fig. 5.3). Sequence
conservation could be of advantage in the case of interspe-
cies communication, such as in non-self-competitive in-
teraction for space and nutrients and hyphal interference,
a phenomenon in which one species suppresses another by
causing its hyphal death (Ikediugwu and Webster 1970). A
foreign species might be attracted to the killing species
(Kemp 1975) and hyphal interference might or might not
include fusion between two species (Ikediugwu 1976; Tra-
quair and McKeen 1977; Boddy 2000). There is certainly
84 Ursula Kües and Mónica Navarro-González

Coprinopsis cinerea
MDSFTT..ITSFTNKPS.........VEVPSDLETGGGSGNGAYCVIA
MDSFTTTAITSFTNQPS.........VEVPSDLETGGGSGNGAYCVIA
MDSFTTIN....................IPTNFETGGGSGNGAYCVIA
MDSFT.LF..SNNVSAT.........TSVPAEEETGGGRGTGAYCTIA
MDFFTTIFASQVADLEP.........VSVPAEEETGGGRGTGAYCTIA

Laccaria bicolor
MDAFTFYITSVD.AIPIDEKTEV...ESLPTD.EESGGSGNNAYCVIA
MDSFTTYFSLAAKAEDISSQ........APVD.EESGGSGNNAYCVIA
MDSFATLSIFFA.AVSGPSD........IPTNADD.GGSGNNAYCVVA
MDSFSTISTSTD................IPTN.YEGGGSGNNAYCVIA
MDSFATLAIFFA.SVSGSSD........IPTNADD.GGSGNNAYCVVA

Cryptococcus neoformans
QSP1 M.SFTTLFTAALVLIAPALVAAAPAAEPAPSVKSQNFGAPGGAYPW
QSP2 M.SFTTLFTAALVLIAPALVAAAPAAEPAPSVKSQNFGAPGGASPI
QSP3 M.SFTTLFTAALVLIAPALVAAAPAAEPAPSVKSQNFGAPGGGKY

Fig. 5.3. Small peptides as possible messenger molecules
in communication of basidiomycetes: Precursor sequences
for a selection of small pheromone-like peptides of Copri-
nopsis cinerea and Laccaria bicolor are given from a mul-
titude of similar small secreted peptides that appear to be
encoded in the species’ genomes (from Niculita-Hirzel
et al. 2008) and precursor sequences from similar small
secreted molecules from Cryptococcus neoformans of
which QSP1 was documented to have quorum sensing
function (Lee et al. 2007). In all cases, the (proposed)
sequence of the mature peptides is shown in bold and
putative processing and modification sides are underlined

room in these interesting antagonistic phenomenons for
communication via pheromone-like molecules.
D. Communication in Population Growth
Control and Germination
The families of pheromone-like peptides of
C. cinerea and L. bicolor and their precursors
have a sequence which somewhat resembles that
of a family of small 10-mer and 11-mer peptides
from C. neoformans (QSP1, QSP2, QSP3) that are
generated from alternatively spliced transcripts of
the gene CQS1 (Fig. 5.3).
The predicted precursors of the families of small peptides of
C. cinerea (33 different genes) and L. bicolor (45 different
genes) have the typical structure of lipo-peptide pheromones
encoded by respective mating type genes in the basidiomy-
cetes: the precursors have an N-terminal signal sequence for
protein secretion, there is an internal charged motif for
putative processing by a endoprotease and at the C-terminal
end, there is the typical CAAX (cysteine – aliphatic amino
acid – aliphatic amino acid – any amino acid) motif for post-
translational processing (Niculita-Hirzel et al. 2008). The
precursors of the C. neoformans peptides have a similar N-
terminal secretion signal and are internally processed at an
analogous location but they do not have the typical CAAX-
motif of fungal pheromone-precursors (Lee et al. 2007).
Most interestingly, without the 11-mer peptide QSP1
the basidiomycetous yeast is unable to grow at low
cell densities. QSP1 apparently mediates a density-
dependent growth phenotype which is reminiscent
of bacterial quorum sensing (Lee et al. 2007).
The word quorum refers in politics to the minimum number
of individuals to undergo a valid decision. In biology, quo-
rum sensing (QS) is understood as cell-cell communication
systems in species of prokaryotes that aid a concerted popu-
lation response. QS as described in bacteria occurs in
growth-phase- and cell-density-dependent manner. Popula-
tions of bacterial species are able to monitor cell density
before expressing a defined phenotype. To this end, they
make use of small specific diffusible signalling molecules
(Whitehead et al. 2001; Daniels et al. 2004; Waters and
Bassler 2005). More recent work suggests that bacterial QS
has also capacity in interspecies and even in cross-kingdom
communication (Lowery et al. 2008; see below).
QSP1 of C. neoformans is the only so far known
fungal autoregulatory signalling molecule for den-
sity-dependent growth control on a peptide-basis.
Autoregulatory signalling molecules of growth
control might act positive as in case of QSP1 in
C. neoformans but there can be others with nega-
tive effects. In the Saccharomycetales yeast Candi-
da albicans, the phenolic alcohol tyrosol acts as
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels
a QS molecule for promoting induction of germ-
tube formation on the yeast cells whilst another
QS molecule, the acyclic sesquiterpene alcohol
farnesol, inhibits this process in favour of typical
yeast growth (Chen et al. 2004; see also Sect. II.E).
Depending on growth medium and yeast spe-
cies, mathematical models deduced from experi-
mental data suggest that reaching constant cell
densities in cultures of unicellular yeasts can be
the result of QS mediated by autoregulators
(Walther et al. 2004). On agar medium, there is
intracolony cell–cell communication as well as
intercolony signalling in various ascomycetes
yeast species, including the baker’s yeast Saccha-
romyces cerevisiae mediated by the volatile alka-
line compound ammonia. Ammonia production
is oriented toward neighbouring colonies growing
in close vicinity and results in growth inhibition
(Palková et al. 1997; Gori et al. 2007). Via such
communication, yeast colonies are able to syn-
chronize with each other in physiology and devel-
opment (Palková and Forstová 2000; Palková and
Vachová 2003, 2006). In the human pathogenic
yeast Histoplasma capsulatum, a-(1,3)-glucan
incorporation in the cell wall occurs growth-de-
pendent at high cell densities and this is under
control of an unknown QS molecule (Kügler et al.
2000). Colonisation on agar medium of this fun-
gus occurs only at a density >106 yeast cells/plate
(Pine 1995). Iron siderophores and hydroxamic
acid released by the yeast cells, acting as growth
factors, regulate this inoculum size effect (Burt
et al. 1981; Burt 1982).
Siderophores sequester required iron from the medium for
the fungus and appear to counteract impurities of the agar
medium. A certain concentration of the siderophores is
required before the mechanism becomes effective – if the
cell density of H. capsulatum is too low this concentration
will not be reached but certain additions to the medium
can overcome the inoculum size effect (Worsham and Gold-
man 1988; Woods et al. 1998). It is thus debatable whether
such a cell-density-dependent mechanism as found in
H. capsulatum is cellular communication in the strictest
sense involving a sender and a receiver of a message. The
case of C. cryptococcus appears to be different from this
since it is medium-independent and unaffected by addi-
tions to the medium. The 11-mer peptide QSP1 might
therefore be a true messenger molecule acting between
cells (Lee et al. 2007).
Whilst most of the so far discussed cases of com-
munication between fungal cells occur in growing
vegetative cultures, growth can also be influenced
by systems of QS at the stage of spore germination.
85
Many species produce self-inhibitors or autoinhi-
bitors of germination when spores are present in
high densities in the environment. Production of
self-inhibitors of spore germination has been
reported for more than 60 different species. Vari-
ous types of secondary metabolites have been
detected to exhibit self-inhibitor functions. Patho-
gens thereby appear to be more specific in the
chemical compounds used than saprotrophs
(reviewed in further detail by Ugalde 2006). For
example, the basidiomycetous rusts Uromyces
phaseoli and Puccinia graminis produce the aro-
matic substances methyl-cis-3,4-dimethoxycin-
nate and methyl-cis-ferulate, respectively (Allen
1972) and mitosporic Colletotrichum species have
been shown to generate one or more self-inhibitors
being such diverse organic compounds as mycos-
porine-alanine and the indol derivatives (E)- and
(Z)-ethylidene-1,3-dihydroindol-2-one, colleto-
fragarone B, (2R)-(3-indoyl)propionic acid, and
(2R)-(3-indolyl) propionic acid (Leite and Nichol-
son 1992; Tsurushima et al. 1995; Inoue et al.
1996). Such very specific chemicals are likely pro-
duced during spore formation to be incorporated
into the spore cell walls or the surrounding muci-
lage (Young and Patterson 1982) from which they
later will successively be released (Ugalde 2006).
Other effective self-inhibitors of germination
produced by conidia likely by fatty acid degrada-
tion are the volatile organic compounds (VOCs)
types of C8-compounds, most often 1-octen-3-ol
(Garrett and Robinson 1969; Chitarra et al. 2004).
1-Octen-3-ol in Penicillium paneum was shown to
render membrane properties in suppressed spores
and to change their internal pH (Chitarra et al.
2005). A similar outcome on cell membranes and
internal pH of fungal spores was postulated for
nonanoic acid (Breeuwer et al. 1997).
Studies in the higher basidiomycetes show
that there can also be communication in spore
populations for stimulation of germination.
In the button mushroom Agaricus bisporus the
volatile isovaleric acid was identified to be an
autoinducer of basidiospore germination (Rast
and Stäuble 1970), which however can be counter-
acted by self-inhibition through CO2 produced by
the spores (Rast et al. 1979). Amongst other un-
known germination-inducing substances pro-
duced by the mycelium of the ectomycorrhizal
species Tricholoma robustum, gluconic acid is in-
ducing germination of its basidiospores (Iwase
1991). n-Butyric acid and related compounds are
86 Ursula Kües and Mónica Navarro-González
operative for Tricholoma matsutake (Ohta 1988).
Volatile germination inducing factors acting in a
species-dependent manner on basidiospores are
also emitted from the mycelia of other basidiomy-
cetes, e.g. from the ectomycorrhizal fungus Lecci-
nium aurantiacum (Bjurman and Fries 1984).
As much as tested, volatiles emitted from mycelia of
related species tend not to act in basidiospore germination
in ectomycorrhizal species (Iwase 1992; Bjurman and Fries
1984). However, basidiospore germination can be trig-
gered by exudates from non-ectomycorrhizal species
such as the basidiomycetous yeast Rhodotorula glutinis
(Fries 1978) or the filamentous ascomycete Ceratocystis
fagacearum (Oort 1974).
E. Communication in Dimorphism
and Asexual Reproduction
There is a growing body of evidence that QS
occurs widespread in dimorphic fungi in order
to positively or negatively regulate switching be-
tween morphological growth forms (Hogan 2006;
Nickerson et al. 2006; Sprague and Winans 2006).
For example, during growth at high cell densities
(>106 cells/ml) of the dimorphic opportunistic
human pathogen C. albicans, farnesol accumu-
lates in the media and, in a QS manner, blocks
the morphological transition from the yeast to the
mycelial stage and with it biofilm formation
(Hornby et al. 2001; Alem et al. 2006; see also
Sect. II.D). Yeast–mycelium dimorphism in Cera-
tocystis ulmi (which causes Dutch elm disease) is
regulated by an unknown QS factor that is not
farnesol (Hornby et al. 2004) but very likely an-
other oxygenated lipid molecule (Jensen et al.
1992). In some strains of the baker’s yeast S. cer-
evisiae, colony density can negatively influence
invasiveness into agar medium and pseudohyphal
growth (Lucaccioni et al. 2007). Autosignalling
aromatic alcohols (phenylethanol, tryptophol)
were shown to suppress filamentous growth in
strains having an active FLO11-flocculation gene
(Chen and Fink 2006). Obviously, different chem-
ical compounds are used by the various yeasts to
regulate dimorphic morphological changes.
In the filamentous ascomycete Aspergillus
niger, the oxygenated lipid farnesol is shown to
suppress conidiation (Lorek et al. 2008). Farnesol
appears not to be produced by A. nidulans but,
when present in the environment, it induces apo-
ptosis-related morphological changes (Semighini
et al. 2006). In the related A. flavus, the formation
of conidia and sclerotia is oppositely density-
regulated. Sclerotia form at low cell density
and conidia at high cell density and different
autoregulatory factors seem to control these
effects. Lipoxygenases appear to be involved in
the production of the factors (Brown et al. 2008).
Lipoxygenases act in the synthesis of fatty acid metabolites
by catalysing the hydroperoxidation of polyunsaturated
fatty acids. Hydroperoxy fatty acids are then converted
into a variety of degradation products, many of which can
act as signalling compounds. Derivatives of fatty acids and
their esters obtained by oxidation through lipoxygenase are
known as oxylipins (Grechkin 1998; Liavonchanka and
Feussner 2006). In fungi, oxylipins and other bioactive
lipid metabolites are shown to affect development, QS, and
effecter molecule production in interaction with other
organisms (Erb-Downward and Huffnagle 2006).
A typical fungal oxylipin is the volatile 1-octen-3-
ol, mediator of the characteristic mushroom
odour (Combet et al. 2006). 1-Octen-3-ol is likely
produced in the fungi from linoleic acid through
oxidation by lipoxygenase into 8(E),12(Z),10(S)-
10-hydroxy-8,12-octadecadienoic acid that subse-
quently is cleaved to 3(R)-1-octen-3-ol and 8(E)-
10-oxo-8-decenoic acid (Wurzenberger and
Grosch 1984; Combet et al. 2006). 1-Octen-3-ol is
a potent and often used signalling molecule in the
fungi (see also Sects. III.B, VI). Application of the
VOCs 1-octen-3-ol and its analogues 3-octanol
and 3-octanone can overcome light-dependence
in conidiation in Trichoderma spp. Sporulating
colonies produce these compounds themselves
and the VOCs mediate as signalling molecules, in
minute concentrations, intercolony communica-
tion for the induction of sporulation on other
colonies from the same or other Trichoderma spe-
cies (Nemčovič et al. 2008).
Other oxylipins are the well studied endoge-
nous precious sexual induction (psi) factors influ-
encing conidiation in A. nidulans by governing
the balance between asexual and sexual reproduc-
tion (reviewed by Ugalde 2006). psiAa [lactone of 5
(S),8(R)-dihydroxy-9(Z),12(Z)-octadecadienoic
acid] induces conidiation and, in turn, counteracts
sexual sporulation. psiBa [8(R)-hydroxy-9(Z), 12
(Z)-octadecadienoic acid] and psiCa [5(S),8(R)-
dihydroxy-9(Z),12(Z)-octadecadienoic acid] are
sporulation hormones that inbibit conidiation
and stimulate ascospore formation (Champe et al.
1987; Calvo et al. 1999, 2001; Tsitsigiannis et al.
2004a, b, 2005; see also Sect. III.B). Moreover,
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels
a defect in production of the oxylipin 10(R)-
hydroxy-8(E),12(Z)-hydroperoxyoctadecadienoic
acid by the human pathogen Aspergillus fumiga-
tus leads to a decrease in conidia production; the
conidia have an altered size, shape and germina-
tion behaviour and, in addition, during fungal
infestation in the human host there is an
increased spore uptake and killing by primary
alveolar macrophages (Dagenais et al. 2008).
Another important group of chemical com-
pounds to be mentioned with respect to regulation
of asexual development in filamentous ascomy-
cetes are terpenes (Ugalde 2006). The best under-
stood molecule is the diterpene conidiogenone
from Penicillium notatum. Conidiogenone is pro-
duced throughout the whole growth phase of the
fungus but when growing in submerged culture,
the local conidiogenone concentration remains
below a threshold required for sporulation. In
contrast, when the fungus enters the aerial
phase, conidiogenone is believed to accumulate
on the hyphal cell walls at concentrations above
threshold levels (>350 pM) so that sporulation
can happen. In liquid culture, thresholds can be
reduced fivefold by increasing Ca2+ concentra-
tions with the consequence that sporulation is
facilitated under submerged conditions (Roncal
et al. 2002a, b; Ugalde 2006).
III. Communication Between Fungi
in Sexual Interactions
A. Communication in Mating
A mating reaction is a prerequisite step in sexual
reproduction of heterothallic fungi. Mating
involves fusion of fungal cells of different mating
type and, in many instances prior to that, a court-
ship period of communication between the cells of
different mating type. Courtship for mating in
ascomycetes and basidiomycetes involves mat-
ing-type specific pheromones, small peptides,
and their also mating-type-specific seven-trans-
membrane G protein-coupled receptors.
Selective attraction between cells of opposite mating type
has been observed for many yeasts and yeast-like species,
both from the ascomycetes and from the basidiomycetes
(see e.g. Bauch 1932; Bandoni 1965; Abe et al. 1975; Flegel
1981; Chenevert et al. 1994; Nielsen and Davey 1995;
Snetselaar et al. 1996; Shen et al. 2002; Lockhart et al.
87
2003). Pheromones functioning in a mating-type-dependent
manner have been isolated from various ascomycetous and
basidiomycetous yeasts. Application of such pheromones to
cells of opposite mating types induces an elongation of yeast
cells directed towards the pheromone source and conjuga-
tion tube formation (Duntze et al. 1970; Imai and Yamamoto
1984; Wong and Wells 1985; Spellig et al. 1994; see works
cited by Flegel 1981; Hiscock and Kües 1999).
In the filamentous ascomycetes, chemical attraction by mat-
ing-type pheromones is observed at fertilization when donor
cells of nuclei (either spores or hyphae) are attracted and fuse
with trichogynes, specialized hyphae differentiated in a my-
celium of an opposite mating type from the ascogonia (the
female reproductive structures) to act as receptors of the
nuclei coming from the male-operating donor cells (Bistis
1983, 1998; Turina et al. 2003; Kim and Borkovich 2004, 2006;
Coppin et al. 2005).
The situation differs from that in the filamentous higher
basidiomycetes, where a reaction by secreted pheromones
prior to hyphal fusion of mating-type competent mono-
karyons was never possible to verify (see Sect. II.C). Mat-
ing of two compatible monokaryons in the higher
basidiomycetes leads to a dikaryon morphologically char-
acterized by the clamp cells formed at the hyphal septa
during the developmental course of nuclear and cellular
division (Kües 2000; Casselton and Challen 2006). The
arch-shaped clamp cells branch from the apical cell of
hyphae, grow backwards, and appear to induce formation
of a peg at the tip of the sub-apical cell to which the clamp
cell eventually fuse (Buller 1931; Badalyan et al. 2004).
Fusion of the clamp cells to the sub-apical cell is B-mat-
ing-type dependent. It is assumed but not proven that at
least in this reaction the mating type pheromones mediate
the communication between the clamp cell and the sub-
apical cell and its peg, respectively (Brown and Casselton
2001; Badalyan et al. 2004).
Expression of pheromones and pheromone recep-
tors in the bipolar ascomycetes is controlled by
the mating type loci encoding types of transcrip-
tion factors (for recent reviews, see e.g. Debuchy
and Turgeon 2006; Tsong et al. 2007; Turgeon and
Debuchy 2007). In the basidiomycetes in contrast,
the pheromone and pheromone receptor genes
are found included with genes for transcription
factors within the single mating type locus of the
bipolar species or, in the tetrapolar species, they
are found in a second mating type locus separate
from the mating type locus containing the tran-
scription factor genes (see the recent reviews by
Casselton and Challen 2006; Bakkeren and Kron-
stad 2007; Casselton and Kües 2007). In either
way, cell-type specific communication systems
88 Ursula Kües and Mónica Navarro-González
are verified so that one mating type produces
pheromones that by a distinctive amino acid
sequence are only recognized by receptors of
another mating type-specificity, which in turn
are also characterized by unique amino-acid
sequences. More information on this can be
found in the above-cited reviews and in various
further chapters in the book Sex in Fungi edited
by Heitman et al. (2007).
In the Mucoromycotina (formerly classified
within zygomycetes, now within the Fungi incertae
sedis; Hibbett et al. 2007), there is a another mech-
anism of communication between cells of different
mating type: mating type recognition and regula-
tion of sexual reproduction (steps zygophore
formation and zygotropism between such struc-
tures of different mating type) is governed by the
b-carotene derivate trisporic acid as the major sex
pheromone and its biosynthetic precursors. The
biosynthesis of trisporic acid is shared between
the two mating types (+) and () of a species. In
short, 4-dihydrotrisporin obtained in both mating
types from b-carotene in the (+) strains are con-
verted to 4-dihydroxymethyl trisporate and in the
() strains to trisporin. These metabolites are ex-
changed between the mating types upon which
trisporin in the (+) mating type is converted to
trisporol and finally trisporic acid and 4-dihydrox-
ymethyl trisporate in the () mating type to methyl
trisporate. Finally, both trisporol and methyl tris-
porate are transformed to trisporic acid (further
reading in the recent reviews by Schimek and Wös-
temeyer 2006; Wöstemeyer and Schimek 2007).
It is interesting to note that the concerted mecha-
nism of trisporic acid formation is not restricted to
strains of one species but due to the widespread usage
of the trisporic acid system in the Mucoromycotina,
precursors can also be exchanged between different
species (Schimek et al. 2003). Even more, strains of
different species may fuse upon trisporic acid-
regulated mating-type recognition such as the para-
sitic species Parasitella parasitica with Absidia
glauca (Kellner et al. 1993; Schultze et al. 2005).
The first three enzymes for the co-operative biosynthesis
of trisporic acid by Mucoromycotina have recently been
described. As a first step confined to both mating types, b-
carotene is cleaved by a b-carotene oxygenase. The final
product of b-carotene degradation is likely 4-dihydrotris-
porin as the last common precursor in both mating types
(Burmester et al. 2007). An NADP-dependent 4-dihydro-
trisporin-dehydrogenase as a () mating-type-specific en-
zyme transforms 4-dihydrotrisporin to trisporin which is
released to reach the complementary mating partner. 4-
Dihydromethyltrisporate dehydrogenase converts 4-dihy-
droxymethyl trisporate, a product of the (+) mating type,
to methyl trisporate. Although both mating types carry the
genes for the latter two enzymes and both transcribe the
genes, active enzymes are only found in the sexually
stimulated () mating type and they are thus strictly
mating-type-specific (Schimek et al. 2005, Wetzel et al.
2009). Receptors for any of the intermediate compounds
produced in trisporic acid synthesis pathway are not
known (Schimek and Wöstemeyer 2006).
Reproduction in the Chytridiomycota such as
Allomyces macrogynus involves fusion between
motile uniflagellate male and female gametes that
can be produced on the same mycelia but in dif-
ferent gametangia. The smaller male () gametes
are sexually attracted to the larger female (+)
gametes by sirenin, a bicyclic sesquiterpene with
a cyclopropane ring, produced by the (+) gametes.
In turn, the () gametes produce a compound
parisin of unknown chemical structure that acts
as attractant of the (+) gametes (Pommerville and
Olson 1987; Pommerville et al. 1988; for detailed
reviews see Gooday 1994; Schimek and Wöste-
meyer 2006; Idnurm et al. 2007).
B. Communication in Fruiting Body
Development
Fruiting bodies with various types of differen-
tiated cells arranged in specific morphological
patterns (Fig. 5.4A, B) are the most complex
structures found within the fungal kingdom (for
reviews see Kües et al. 2004; Pöggeler et al. 2006;
Wösten and Wessels 2006). Fruiting body forma-
tion demands highly co-ordinated cellular pro-
grams of synchronized development and
differentiation. This includes also a co-ordinated
and specific fusion of cells such as occurring
between the outer layer of enveloping hyphae in
the formation of the peridia (outer rind tissue
formed by melanized small and densely arranged
cells) of ascomycetous fruiting bodies (Read
1994). In the early progress of “tissue” differenti-
ation in the initials (nowadays known as second-
ary hyphal knots, i.e. small compact hyphal
aggregates induced by a light signal; Kües et al.
2004) of fruiting bodies of the basidiomycete C.
cinerea, anastomosis occurs frequently between
cells in the core of the structure but not between
the large hyphae of its outer layer (Van der Valk
and Marchant 1978). Some “tissues” in the fully
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels
Fig. 5.4. A, B A hand-cut and a microtome cross-section
through fully developed primordia of Coprinopsis cinerea
directly prior to fruiting body maturation showing the
complex tissue differentiation in the multicellular struc-
ture. A white symbol * marks the plectenchymatic mycelial
regions in the pileus whilst a black symbol * indicates the
position of the gills on which surface the prosenchymal
hymenium is localized. C A young mushroom of the fly
agaric Amanita muscaria (photograph kindly supplied by
developed primordium are plectenchymal such as
the inner parts of the gills and pileus with inter-
woven, multiple branched, and interconnected
hyphae, whilst others such as the hymenium and
subhymenium are prosenchymal with unbran-
ched parallel arranged hyphal cells (Kües 2000;
Fig. 5.4B). Obviously, a blueprint exists for the
fruiting body which determines not only cell
shapes and sizes and places but also which of
the cells will interact by fusion and which not.
To verify this blueprint, communication and co-
ordination between neighbouring cells, respec-
tively between different mycelial areas within the
developing primordium should be required.
In the filamentous ascomycetes, diffusible fac-
tors including the mating-type pheromones can
play a role in fruiting body development such as
in frequency of fruiting body induction, in male-
female communication and fertilization of
the trichogyne, in ascocarp maturation and in as-
cospore formation (reviewed by Dyer et al. 1992;
Pöggeler et al. 2006). In the heterothallic N. crassa
as well as in the homothallic Sordaria macrospora,
mating-type pheromone communication with their
receptors is required for (efficient) fruiting body
maturation (Kim et al. 2002; Mayrhofer et al. 2006),
whilst in the also heterothallic Podospora anserina
the pheromone function is restricted to fertilization
(Coppin et al. 2005). Furthermore, in the homo-
thallic Gibberella zeae the pheromone communica-
tion system is not essential for sexual development
89
U. Helle) that can only develop in symbiosis with a woody
host. D A cross-section through a pine root colonized by
an unidentified ectomycorrhizal basidiomycete (prepara-
tion by E. Fritz, photograph by A. Olbrich). Marked are the
central stele (S), the cortex (C), the Hartig net of fungal
cells (white symbol *) in between plant cortex cells result-
ing in a large surface area for nutrient transfer and com-
munication, and the ectomycorrhizal outer mantle (black
symbol *) that covers the surface of the root
but it can enhance its frequency (Kim et al. 2008;
Lee et al. 2008).
In the homothallic A. nidulans (telemorph
Emericella nidulans) producing its meiospore in
cleistothecia (Pöggeler et al. 2006), the fruiting
body production is determined by the availability
of the already mentioned psi factors. Effects of
the different psi factors on cleisthothecia produc-
tion are opposite to those on conidiation (Tsitsi-
gianni et al. 2004a, b, 2005; for more details see
Sect. II.E). Exogenous application of certain types
of lipids including linoleic acids can influence
fruiting in the species (Champe et al. 1987; Calvo
et al. 1999). Production of perithecia (Pöggeler
et al. 2006) in N. crassa, Nectria haematococca as
in plant pathogenic Ceratocystis species is also
enhanced by addition of linoleic acids, even
when added only in minute amounts (Nukina
et al. 1981; Marshall et al. 1982; Dyer et al. 1993).
The observations indicate that oxylipin control of
ascocarp formation is very likely widely
distributed within the ascomycetes.
VOCs as autoregulatory compounds emitted
from the own mycelium can positively (Basith and
Madelin 1968) or negatively (Moore-Landecker
and Shropshire 1984) influence ascocarp forma-
tion. On the whole, however, information in asco-
mycetes on induction of fruiting, fruiting body
differentiation and maturation by extracellular
morphogens is sparse (Moore-Landecker 1992;
Ugalde 2006; Pöggeler et al. 2006).
90 Ursula Kües and Mónica Navarro-González
Little is yet known on cellular co-ordination of
fruiting body development and maturation in the
higher basidiomycetes (Kües 2000; Walser et al.
2003; Kües et al. 2004; Wösten and Wessels 2006),
let alone on the participation of signalling mole-
cules in this. In C. cinerea, work by Kamada and
Tsuji (1979) showed that a diffusible darkness-
induced factor from the fully developed
primordia caps (Fig. 5.5) can bring about stipe
elongation and fruiting body maturation. Also,
there is a function of the B mating type phero-
mones and their receptors in inducing fruiting
body maturation at the stage of karyogamy once
the primordia were fully established in struc-
ture (Kües et al. 2002). In various instances,
Fig. 5.5. A A slug grazing on tufts of fruiting bodies of
Coprinellus disseminatus grown on a meadow. B–F An
experiment for baiting animals to fresh fungal cultures
with mushrooms: Petri dish cultures of Coprinopsis
cinerea containing fully developed primordia ready to
undergo overnight fruiting body maturation (see
Fig. 5.4A) were moved either with covering lids or with-
out lids at an evening in the late summer (in August) into
a meadow underneath some bushes. Overnight, the fruit-
ing bodies matured and snails and collembolans were
apparently attracted to the cultures for consuming the
fungus. Snails appeared to have consumed the complete
culture with mycelium and fruiting bodies inclusive the
meiotic spores as well as the agar. Faecal pellets (B) left in
the Petri dishes contained hyphal fragments as well as
exogenous addition of surface-active compounds
(lipids, detergents, amphipatic sugars and gly-
colipids, such as cerebrosides and other
membrane-interacting compounds, specific cyto-
lytic membrane-interacting proteins, such as
ostreolysin from Pleurotus ostreatus) stimulated
fruiting body initiation in species of higher basi-
diomycetes although not all such compounds are
active (Kües and Liu 2000; Magae et al. 2005;
Magae and Ohara 2006; Berne et al. 2007, 2008).
From mutant analysis in C. cinerea, lipid-linked
membrane-associated signalling processes are
anticipated in the process of fruiting body initia-
tion (secondary hyphal knot formation) occurring
spatiotemporally defined within mycelial cultures
masses of dark brown-stained basidiospores (C), suggest-
ing that the fruiting bodies were fully developed before
they were consumed. Whilst the lids on closed Petri
dishes hindered larger animals reaching the fungal cul-
tures, larvae and adult stages of collembolans were found
in the cultures either at the underside of the opened
mushroom cap (D see tip of arrow; inset: enlarged view
on the transition between fruiting body gills and the
centred stipe reveals many black-stained faecal pellets
laid onto the mushroom tissues) or within the still pres-
ent vegetative mycelium on the agar (see examples in E;
the arrow in the figure points at a faecal pellet laid by the
adult animal). Also the collembolans consumed the basi-
diospores and loads of intact spores are seen in squeezed
faecal pellets (F,G)
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels
(Liu et al. 2006). Following initiation at multiple
places, a bigger part of young fruiting structures
will be aborted in favour of one or a few, with
translocation of nutrients towards those struc-
tures that eventually will mature as fully devel-
oped fruiting bodies (Madelin 1956; further
discussed by Moore 1998). The concerted and
locally directed action suggests that intra-colony
communication happens.
C. cinerea mutants with defects in fruiting body initiation,
in cap and stipe tissue formation, and in stipe elongation
have been used to characterize responsible genes. In this
way, four enzyme encoding genes were identified:
1. cfs1 for a cyclopropane fatty acid synthase with a func-
tion in initiation of fruiting body development (Liu
et al. 2006)
2. eln2 for a new type of cytochrome P450 acting in stipe
elongation and being distantly related to the oxidore-
ductases from A. flavus and A. parasiticus which con-
vert O-methylsterigmatocystin to aflatoxin (Kües 2000;
Muraguchi and Kamada 2000)
3. ich1 for an O-methyltransferase acting in pileus differ-
entiation and being distantly related to enzymes of A.
flavus and A. parasiticus which are involved in the
conversion of sterigmatocystin to O-methylsterigmato-
cystin and dihydrosterigmatocystin to dihydro-O-
methylsterigmatocystin (Muraguchi and Kamada
1998; Kües 2000)
4. eln3 for a potential glycosyltransferase probably
involved in cell wall biogenesis (Arima et al. 2004).
For none of these enzymes are the substrates yet known, but
the possibility of producing signalling molecules for devel-
opment and communication has been discussed for the first
three enzymes (Kües 2000). In this connection it is interest-
ing to note that, in the Aspergillus species, aflatoxin produc-
tion is tightly linked to conidiation although it is not
essential for sporulation. Block of aflatoxin synthesis affects
however the numbers of conidia that will be produced
(Calvo et al. 2002).
In A. bisporus, the oxylipin (8E)-10-oxo-8-
decenoic acid appears to stimulate stipe elongation
as well as mycelial growth and fruiting initiation
(Mau et al. 1992; Mau and Beelman 1996). In the
young yet closed mushrooms (button stage 20 mm
diam., with closed caps; medium stage 35 mm
diam., with closed veil), 1-octen-3-ol accumulates
at highest levels (Mau et al. 1993; Cruz et al. 1997),
possibly to function in protection against moulds
(Okull et al. 2003) and bacteria trying to infest the
mushrooms (Beltran-Garcia et al. 1997). General-
ly, the concentration of these and other VOCs
increases dramatically upon mushroom wound-
ing, supporting a role in wound-activated chemi-
cal defence (Wadman et al. 2005; Spiteller 2008).
91
Many other VOCs as potential signalling mole-
cules are produced by mushrooms – a literature
account collected more than 250 different scent
compounds that are either plain hydrocarbons,
heterocycles, alcohols, phenols, acids, and deriva-
tives, together with sulfur-containing molecules
(Chiron and Michelot 2005).
IV. Communication Between Fungi
and Bacteria
Cross-kingdom QS has been encountered between
the gram-negative bacterium Pseudomonas aeru-
ginosa and C. albicans. P. aeruginosa suppresses
the filamentous pathogenic growth form of
C. albicans by secreting its own cell-cell signalling
molecule 3-oxo-C12 homoserine lactone (Hogan
et al. 2004). In turn, farnesol produced by C.
albicans inhibits the swarming motility of the
bacterium (McAlester et al. 2008).
Taking signalling compounds developed for
self QS and using them for inter-kingdom commu-
nication is not the only way how organisms within
the same complex biotop may influence the intra-
species communication of others. Various fungi for
example have evolved defence mechanisms against
bacteria by blocking their QS systems. Within the
genus Penicillium for example, 33 of 50 tested
species were found to produce quorum sensing
inhibitor (QSI) compounds against P. aeruginosa.
The lacton mycotoxin patulin and the antibiotic
penicillic acid are two potent QSI compounds neg-
atively influencing QS-controlled gene expression
in the bacterium (Rasmussen et al. 2005). As an-
other mean of interrupting QS of competitive bac-
teria in the same environment, specific fungi in
forest soils produce a lactonase in order to degrade
N-acyl homoserine lactones used by bacteria in the
rhizosphere as signalling compound (Uroz and
Heinonsalo 2008).
Probably caused by competing for resources,
bacteria can negatively influence growth, devel-
opment and reproduction of fungi by releasing
VOCs. For example, perithecial formation by
Gelasinospora cerealis is inhibited by VOCs emit-
ted by several kinds of bacteria (Moore-Land-
ecker and Stotzky 1972), bacterial VOCs inhibit
the ascogenous system of Pyronema domesticum
(Moore-Landecker 1988), abnormal shortening
of conidiophores of Aspergillus giganteus has
been observed (Moore-Landecker and Stotzky
92 Ursula Kües and Mónica Navarro-González
1973) and bacterial VOCs inhibit fungal spore
germination (Barr 1976). Exudates from bacterial
isolates from sporophores, mycorrhizae or soil in
contrast stimulate germination of spores of ecto-
mycorrhizal fungi (Ali and Jackson 1988). Most
interesting are the mycorrhization helper bacte-
ria acting positively in establishing the symbiosis
between ectomycorrhizal basidiomycetes and
woody plants. Amongst different effects exerted
by the various species of helper bacteria, a Strep-
tomyces strain was shown to produce an auxin-
related compound termed auxofuran that stimu-
lates growth of the fly agaric Amanita muscaria
and suppresses growth of fungal root pathogens
(Frey-Klett et al. 2007; Schrey and Tarkka 2008).
A potent inducer of quinone-character (basidif-
ferquinone) for the induction of fruiting in the
saprotrophic mushroom Flavolus arcularius is
known from another Streptomyces strain
(Azuma et al. 1990).
V. Communication Between Fungi
and Plants
Fungi and plants may undergo symbiotic reac-
tions where both obtain a benefit from the inter-
action or they may exist in interactions where the
selfish fungi live as a pathogen on the plants and
cause damage to them. In both types of fungal life
styles, communication between fungus and plant
can play a crucial role.
The rhizosphere with its multiple root–root,
root–microbe, and microbe–microbe interactions
is a best ecological example for complex interspe-
cies communications (Bais et al. 2004, 2006). Exu-
dates from plant roots for example are shown to
stimulate in a presymbiontic growth phase germi-
nating spores of AM fungi in nutrient uptake
(Bücking et al. 2008). Plant roots may synthesise
certain types of apocarotenoids (e.g. strigolac-
tones) as signalling compounds to the obligate
symbiotic AM fungi in stimulating their growth
by inducing hyphal branching and in the estab-
lishing the endomycorrhizae (Akiyama et al. 2005;
Besserer et al. 2006; Sun et al. 2008). Flavenoids
from root exudates also promote hyphal growth,
differentiation and root colonization by AM fungi
in a genus- and species-specific manner (Steinkell-
ner et al. 2007). Plants in turn receive signals from
the AM fungi – ungerminated and germinated
spores of Gigaspora and Glomus species from
the Glomeromycota release diffusible molecules
that in cells of a host plant such as soybean (Gly-
cine max) are perceived through a Ca2+-mediated
signaling and thereby induce the expression of
AM-related host genes (Navazio et al. 2007b). So
far, the types of the chemical signals released from
AM fungi have not been defined. Communication
of the fungi with their hosts generally divides in
two steps – prior to direct cellular contact and
during the contact. Compounds used in the two
stages of dialogue could indeed differ. Plants for
example chance their flavenoid pattern from pre-
symbiotic stages to root colonization (Genre and
Bonfante 2007; Steinkellner et al. 2007).
Like AM fungi, the presence of non-mycorrhi-
zal fungi in the environment also are sensed by the
plant through intracellular Ca2+ changes upon ex-
posure by secreted fungal molecules. Several path-
ogenic fungi elicit extremely specific variations of
Ca2+ concentration (Lecourieux et al. 2006). Most
interestingly, the intracellular Ca2þ responses on
pathogens such as the nectrotrophic Botrytis
cinerea and on simultaneous presence of biocontrol
fungi such as Trichoderma atroviride are different.
The plants can discriminate between the metabo-
lites of the two different situations by different
kinetics of the Ca2+ responses and by specific pat-
terns of intracellular accumulation of reactive oxy-
gen species (Navazio et al. 2007a). Different types of
elicitors, messenger substances that cause defence
reactions in plants, are known to be produced by
pathogenic fungi attacking roots, leaves, and other
parts of plants as well as by non-pathogens such as
by the Trichoderma biocontrol fungi (for a review
see Ebel and Scheel 1997; Harman et al. 2004; Ber-
rocal-Lobo and Molina 2008). Upon host penetra-
tion, fungi secrete effector molecules into the
apoplast or into host cells for suppressing plant
defence responses. However, these effectors may
then act also as signals to the plants to reinforce
defence (see the recent review by Göhre and Robat-
zek 2008). Mostly, elicitors and effectors are
of proteineous nature or oligo-carbohydrates but
phytotoxic sesquiterpenoids are also described. For
more details, the reader is referred to the respective
cited reviews.
To illustrate the broad variety of fungal elicitors, peptaibol
as non-ribosomally produced peptide (Viterbo et al. 2007),
swollenin as a protein with a carbohydrate-binding do-
main (Brotman et al. 2008), small cysteine-rich proteins of
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels 93

the ceratoplatanin family of proteins (Djonovic et al. 2006; plant in order to proceed in its development.
Vargas et al. 2008), and oligosaccharides released by Tri- Metabolites resulting from the photosynthesis of
choderma-mediated enzymatic degradation of fungal and
plant cell walls (Harman et al. 2004) are named at this
woody plants are transferred to the symbiontic
place as characterized elicitors from Trichoderma species. fungi through interaction between root cells and
the Hartig net (Fig. 5.4D) for general nutrition
(Anderson and Cairney 2007) but this may not
Compared to AM mycorrhiza, even less is known
be all what is required from the host for fruiting
about communication between ectomycorrhizal
body induction of the fungus. Otherwise, it should
fungal species and their plant hosts (Martin et al.
have been possible to produce mushrooms in the
2001, 2008). For the truffle Tuber borchii–Tilia
laboratory under good nutritional conditions
americana confrontation, 29 different VOCs specif-
without the plant host (Kües et al. 2007). As dis-
ic to the premycorrhizal stage have been encoun-
cussed in Sect. III.B, fruiting body development in
tered as candidates mediating communication with
saprotrophs is stimulated by surface-active com-
its host roots over a distance of a few centimetres.
pounds. There might be similar effects by specific
Amongst these are e.g. the sesquiterpenes germa-
plant metabolites on fruiting of ectomycorrhizal
crene D, dehydroaromadendrene, b-cubebene, and
species.
longicyclene known to be produced by plants
Plant and fungi communicate with each other
(Menotta et al. 2004). The particular VOCs emitted
also above direct biotrophic interactions. Plant
by the ascomycete T. borchii to mediate commu-
VOCs such as methyl salicylate for example trig-
nications with its host plants are yet to be estab-
gers sporulation of entomopathogenic fungi
lished. However, 1-octen-3-ol and trans-2-octenal
(Hountondji et al. 2005, 2006) and plant oxilipins
from the fungal volatiles were shown to induce an
can replace endogenous oxylipins in control of
oxidative burst of H2O2 in the non-host Arabidop-
growth and development in Aspergillus species
sis thaliana and they inhibited development of the
(Mita et al. 2007; Brodhagen et al. 2008). Most
plant (Splivallo et al. 2007b). In nature, the burnt
fascinating are cases of hijacking plant signals
phenomenon referring to a zone of very meager
addressed to pollinators by rust fungi for their
vegetation around trees with roots colonized by the
spore distribution by insects (Kaiser 2006). Mus-
truffle may relate to this – some of the manifold
cid and anthomyiid flies and halictid bees were
VOCs emitted from the truffles (or from the bacte-
shown to be attracted to phenylacetaldehyde, 2-
ria commonly growing within the tubers) may
phenylethanol, benzaldehyde, and methylbenzo-
exert phytotoxic effects on seed germination and
ate scents of pseudo-flowers induced by Puccinia
reduce root growth of the vegetation (Splivallo
monoica on crucifer hosts (Roy and Raguso 1997;
2008; Tarkka and Piechulla 2008).
Raguso and Roy 1998).
Plants in biotrophic interactions may send out
signals in turn to the fungi. The rust fungus Uro-
myces fabae stimulates volatile production by host
bean plants. Of these VOCs, nonanal, decanal, and VI. Communication Between Fungi
hexenyl acetate promote haustoria formation of and Animals
the pathogen, whilst the terpenoid farnesyl acetate
counteracts the effects by the VOCs (Mendgen
Interactions between fungi and animals may serve
et al. 2006).
different functions: the fungi may use animals as
In the case of ectomycorrhizal basidiomy-
vectors for their distribution or for their nutrition,
cetes, root exudates from the woody hosts stimu-
and the animals may enjoy the fungi as a resource
late germination of the poorly germinable
of food for their living or use them as indicators
basidiospores (Fries 1989; Ishida et al. 2008) and
for the availability of other food sources or also as
certain flavonoids from root exudates (hesperidin,
indicators of places for breeding. Depending on
morin, rutin, quercitrin, naringenin, genistein,
the target, a fungus may send out signals for
chrysin) are nominated as candidates for this in-
attraction or for repellence of animals. Usually,
teraction (Kikuchi et al. 2007). The production of
the infochemicals in fungus–animal communica-
fruiting bodies of ectomycorrhizal species
tion are of a volatile and mostly organic nature
(Fig. 5.4C) is another example of fungus–plant
(Thakeow et al. 2007). This is because fungi tend
communication where the fungus requires the
to be sessile whereas animals are commonly
94 Ursula Kües and Mónica Navarro-González
motile. Moreover, the large variety of different
VOCs that can be formed by living cells offers
multiple possibilities to develop and use very spe-
cific and discriminative chemical languages be-
tween interacting organisms according to need
in a much more complex environment.
To give an example of how a sessile fungus takes advantage
of mobile animals in an apparent symbiontic interaction,
ascospores of the heterothallic grass endophyte Epichloë
typhina are distributed by females of the fly Brachypodium
sylvaticum that oviposit on the fungus (Bultman et al.
1998). Of the various volatiles produced simultaneously
either by the fungus, its plant host, or by them collectively,
the fungitoxic sesquiterpene alcohol chokol K of Epichlöe
species and the methylester methyl (Z)-3-methyldodec-2-
enoate have been identified to be specific attractors of the
female flies (Steinebrunner et al. 2008a, c). It is interesting
to note that chokol K has a dual role for the fungus: one is
attracting flies for spore distribution, another to repress
growth of other fungi on the plant (Schiestl et al. 2006;
Steinebrunner et al. 2008b).
Fungi may communicate with all types of animals
up to the humans. Molds for example cause a
damp-musty or an earthy-raunchy smell unpleas-
ant for humans, resulting in avoidance reactions
and refusal of infested food (Schnurer et al. 1999;
Kuhn and Ghannoum 2003), whilst aromas from
edible mushroom can be very attractive to
humans (Chiron and Michelot 2006). Mycophagy
within mammals is widely distributed and often
they have a possible preference for hypogeal spe-
cies such as truffles and false truffles, species that
rely on mammals for spore distribution (Claridge
and May 1994; Johnson 1996; Wheatley 2007).
Strong and special aromas make them traceable
for mammals below the covering soil (Gioacchini
et al. 2005, 2008). Mycophagy is also found within
mollusca (Silliman and Newell 2003; Fig. 5.5A),
earthworms (Moody et al. 1996), nematodes
(Mikola and Sulkava 2001), and various types of
arthropods (Maraun et al. 2003; Fig. 5.5D, E; see
below), combined with spore distribution by ani-
mal faeces and, for some fungal species, also with
a positive effect on germination (see Fig. 5.5B, C,
F, G and the cited papers).
Snails have been found to be attracted to fungi likely by
their odours (Fig. 5.5A) but the nature of the VOCs caus-
ing their reaction needs to be elucidated. Certain mush-
rooms such as Clitopilus prunulus and Clitocybe faccida
produce VOCs (e.g. 1-octene-3-ol, clitolactone) that pro-
tect them from being eaten by slugs (Wood et al. 2001,
2004). When being attracted to and consuming
mushrooms, the snails also eat the spores which, together
with undigested parts of hyphal filaments, are excreted
with the faeces (Fig. 5.5B, C; Navarro-González, unpub-
lished data). Currently, it remains to be shown whether
spores and/or hyphal fragments from the mushrooms in
faeces of snails will grow. However, faeces of the snail
Littoraria irrorata grazing on fungal mycelium on infested
cordgrass Spartina alterniflora are filled with hyphal frag-
ments that appear able to grow. The snail distributes the
faeces with fungi primarily of the ascomycetous genera
Phaeosphaeria and Mycosphaerella onto new blades of
cordgrass, where the fungi invade the cordgrass through
wounds that the snails created purposely in the leafs
(Silliman and Newell 2003).
Communication is best analysed between fungi
and arthropods. Often it is 1-octen-3-ol as part of
the typical mushroom odour that is used to specif-
ically attract insects to mushrooms, such as the
mycophagus beetle Cis boleti to the fruiting bodies
of Trametes gibbosa (Thakeow 2008; Thakeow
et al. 2008) and various wood-living beetles and
the moth Epinotia tedella to fruiting bodies of
wood-decaying Polypores (Fäldt et al. 1999). Strik-
ingly, in case of the beetles it is preferentially again
the females that are attracted by the fungal odour
(Fäldt et al. 1999; Thakeow et al. 2008). This app-
ears also be the case for the truffle beetle Leiodes
cinnamomea: obviously only the females are attra-
cted in a yet unknown way to Tuber melanosporum
fruiting bodies, whilst the male insects are not attr-
acted by the fungus but secondarily by the females
infesting the truffles (Hochberg et al. 2003).
Fungal odours can also show the way for pre-
dators – the combination of 1-octen-3-ol and
octan-3-none acts as a magnet for the predator
on fungus-insects Lordithon lunulatus (Fäldt et al.
1999). In other instances, 1-octen-3-ol in combi-
nation with methyl cinnamate repel the mycopha-
gus collembolan Proisotoma minuta and the
production of these VOCs by T. matsutake pro-
tects its mushrooms against P. minuta (Sawahata
et al. 2008). On its own as pure compound,
1-octen-3-ol acts as deterrent to both sexes of the
phorid fly Megaselia halteria, a pest in the culti-
vation of the edible A. bisporus (Pfeil and Mamma
1993), whilst in combination with other C8 com-
pounds (3-octanone, 1-octen-3-one) it may attract
females to the mushroom culturing beds (Grove
and Blight 1983).
Mites feared by mycological laboratories as pests in fungal
cultures also use the aliphatic alcohol 1-octen-3-ol and
related C8 compounds as signalling molecules to localize
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels
the emitting fungi in order to feed on them (van Haelen
et al. 1978; Chaisaena 2008; Fig. 5.6); and a case where a
nematode was attracted by B. cinerea through 1-octen-3-ol
as well as 3-octanol has also been documented (Matsumori
et al. 1989). In contrast, the banana slug Ariolimax colum-
bianus reacts deterred when confronted with 1-octen-3-ol
(Wood et al. 2001). Future studies need to reveal how
much it is also a matter of concentration whether the
different organisms sense 1-octen-3-ol as appealing or
repellent.
Mushrooms of different species all have their own
special aroma composed of different combinations
of VOCs from chemical categories such as alco-
hols, aldehydes, ketones, and isoprenoids (e.g. see
Mau et al. 1997; Venkateshwarlu et al. 1999; Bous-
tie et al. 2005; Wu et al. 2005; Splivallo et al. 2007a;
Thakeow 2008; see the review by Chiron and
Michelot 2005). Therefore probably not surpris-
ingly mushroom–insect interactions can be very
specific, particularly for fresh mushrooms, where-
as this is found not so much for decaying fruiting
bodies (Guevara et al. 2000; Jonsell and Nordlan-
der 2004; Orledge and Reynolds 2005). It is possi-
bly the combination of different volatiles emitted
by fresh specimens that leads to distinct attrac-
tions for insects. In contrast, decaying mushrooms
have less striking VOC differences and become
chemically more similar to each other (Jonsell
and Nordlander 2004; Thakeow et al. 2007).
Fig. 5.6. Dust mites of the species Tyrophagus putrescen-
tiae are easily attracted to fungal cultures in order to
consume the obviously tasty mycelium of the
fungus Coprinopsis cinerea. A Overview and B enlarged
95
A paradigm for successive alterations in
arthropode attraction is presented by the release
of changing VOCs during the course of wood
decay by fungi. Saproxylic insects are attracted
by wood–rotting fungi to the resource wood – as
the fungal wood decay progresses, an alteration
in VOC patterns is detected which tells the
insects the momentary levels of decay and
whether it is at a suitable stage for a given insect
to infest the wood for feeding and/or breeding
(Holighaus and Schütz 2006; Thakeow et al.
2007).
VII. Conclusions
All the time, an ever-changing plethora of second-
ary metabolites augmented by proteinaceous
compounds is found in the biosystems produced
as a sum by the various bacteria, the lower eukar-
yotes such as fungi, the plants, and/or the animals
living in a common habitat – not to forget that
there are in addition also compounds coming
from spontaneous chemical reactions occurring
in the same environment. Specific compounds in
such complex mixtures referred to as infochem-
icals have distinct communication functions with-
in a species and between different species on dual
sector of a C. cinerea culture infested by mites:
Perforations in the mycelial carpet indicate loss of
mycelium by grazing mites. C A mite grazing at the myce-
lium
96 Ursula Kües and Mónica Navarro-González
up to multiple organisms levels. The different
functions might simultaneously come into action.
1-Octen-3-d and mushroom development
may be taken as an example how at the same
time one single compound may be used as a mes-
senger for different targets: in the induction of
mushroom development, in the attraction of ani-
mals for spore distribution, as a repellent of ani-
mals that otherwise might just graze on the
fungus, and as a toxic compound reacting on
mycotrophic moulds and bacteria that might in-
fest and decompose mushrooms for their feed or
that might suppress mushroom development.
Using one and the same compound for all these
different targets happening in parallel suggests a
tight co-evolution between different organisms in
a given biosystem. This reasonable avenue raises
however the question: how does it come that ecol-
ogy does not go wrong in such a complex envi-
ronment if so many different organisms react in
so different way on the same signal? Schiestl et al.
(2006) recently suggested that defence mechan-
isms developed first in evolution and that defence
chemicals were then appointed secondarily into a
function of messenger molecules. To verify the
different functions, one can expect that a fine-
tuning of sensitivity via adaptation to specific
compound concentrations will be very important
and, connected to this, the steepness of the gradi-
ents of signalling molecules being sent out from a
source to a receiver (Matsui 2006; Mita et al.
2007). Particularly the VOCs offer special qualities
to function as messengers since in most instances
they easily move in air and, by that, they are also
very easily removed from a system once they are
no longer required.
To fulfil their functions as infochemicals, the
language of messenger molecules must be shared
by a sender and a receiver. A multitude of chemical
compounds of very different natures has already
been identified acting as infochemicals. In most
instances, signalling molecules are types of second-
ary metabolites. Like 1-octen-3ol, they are not al-
ways very specific to just one or a few combinations
of communicating organisms. Specificity might be
more enhanced with organic compounds of chem-
ically higher complexity, such as for example
sesquiterpenes. Of all, peptides are the most specific,
due to the multitude of possible combinations of
amino acids giving a distinct sequence. Not surpris-
ingly therefore, many of the fungi appoint peptides
in sexual attraction to each other, being one of the
most specific biological tasks to fulfil in ecology and
evolution.
Currently, we are only at the beginning of
perceiving how complex chemical communication
is in nature, both on the level of individuals and,
generally, on the whole community level. We do
not suggest that our literature overview is com-
plete, neither in the examples where communica-
tion with a fungus plays a role nor in the type of
messenger molecules used – there are too many
individual observations reported which are often
unlinked to each other and which often are not
deeply enough evaluated for judging whether they
present a true communication system. One
reason for this is that it is difficult to consider
the complete complexity within an ecosystem.
Furthermore, too often evidence for the actions
of signalling molecules is still only circumstantial.
Better chemical definitions of signalling com-
pounds and their production and better defini-
tions of their concentration spectra of activity
are a prerequisite for understanding the various
chemical languages fungi may use in communica-
tion with their environments.
With the apparent exception of the mating
systems using diffusible peptide pheromones
(see above-cited reviews on fungal mating type
systems), the way that signals are perceived by
the receiver and the subsequent intracellular sig-
nalling pathways within the sensitized cells are
in most instances fully unclear (Ugalde 2006).
Establishing specific receptors for chemical com-
munication and linking this to intracellular com-
munication (signalling) cascades is part of
evolving a chemical language in environments in
which naturally a high noise of different chemical
compounds exists. Elucidating these will be the
exigent task of future work in molecular and cel-
lular biology on the road to unravelling the vari-
ous mechanisms of communication systems that
exist in nature. A very exciting question remains
for theoretical biologists and behavioural scien-
tists (understood in a broader sense than in the
usual limitation to animals) to go more deeply
into the ecological and evolutionary functions
and consequences of communication systems, in-
cluding those in which fungi participate.
Acknowledgements The authors are very grateful to
U. Helle, A. Olbrich, and E. Fritz for supplying the photos
on ectomycorrhizal fungi. Research in our laboratory on
fungal volatiles and on mites is done in collaboration with
P. Thakeow, P. Plašil, and S. Schütz from the Division
 Communication of Fungi on Individual, Species, Kingdom, and Above Kingdom Levels
Forest Zoology and Forest Conservation at the Büsgen-
Institute Göttingen, and research on mating type genes in
collaboration with international consortia on analysing
genomes of higher basidiomycetes supplied by the DOE
Joint Genome Institute in Walnut Creek, California.
Research on L. bicolor is done within the framework of
the Evoltree programme of the European Community.
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6 Yeast Killer Toxins: Fundamentals and Applications

FRIEDHELM MEINHARDT1, ROLAND KLASSEN1

CONTENTS with the discovery of a so-called killer strain of the

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 brewer’s yeast Saccharomyces cerevisiae (Bevan and
II. Chromosomally Encoded Killer Toxins . . . . . . . 107 Makower 1963). At present, we are aware of a pano-
A. Williopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 ply of killer toxins originating from both ascomyce-
B. Pichia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 tous and basidiomycetous yeasts. Toxin sizes range
C. Kluyveromyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
III. Extrachromosomally Encoded Toxins . . . . . . . . . 112 from small chromosomally encoded polypeptides to
A. dsRNA Virus Toxins . . . . . . . . . . . . . . . . . . . . . . . . 112 larger di- or trimeric protein complexes, the latter
1. K1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 being encoded by extranuclear DNA or RNA ele-
2. K28 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 ments of viral origin which parasitize the host cell’s
3. Other dsRNA Virus Toxins . . . . . . . . . . . . 116 cytoplasm. Continually growing understanding of
B. Linear Plasmid-Encoded Toxins . . . . . . . . . . . 116
1. Group I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 the mode of action of various killer toxins along
2. Group II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 with the corresponding immunity mechanisms not
IV. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 only facilitates the recognition of new toxic principles
A. Antifungals for Human Therapy . . . . . . . . . . . 120 but also significantly contributes to our understand-
B. Antifungals in Agriculture, Food and ing of precarious mechanisms during cell prolifera-
Feed Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
C. Killer Toxins in Biotyping . . . . . . . . . . . . . . . . . . 123 tion. Reviews addressing yeast killer toxins (generally
V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 or more specifically dsRNA- or dsDNA-encoded
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 ones) have been published over the years (Stark
et al. 1990; Bussey 1991; Magliani et al. 1997; Schmitt
and Breinig 2002, 2006; Schaffrath and Meinhardt
2004; Golubev 2006; Klassen and Meinhardt 2007).
I. Introduction However, significant progress in the identification of
toxic principles and strategies, immunity mechanism
and promising approaches for the application of such
Microbes competing for limited resources have
proteins has been made only rather recently. In this
established a broad arsenal of lethal compounds aim-
contribution, we summarize current knowledge on
ing at the inhibition or destruction of competitors.
killer toxins and toxic principles, starting from chro-
Toxic matters released by microbial killers include
mosomally encoded ones, followed by dsRNA- and
classic antibiotics and low/medium molecular weight
dsDNA-encoded toxins and finally we discuss the
substances belonging to various chemical classes,
state of the art of toxin applications.
but – rather frequently – there are antibiotic peptides
and even large protein complexes termed bacterio-
cines (bacteriocidic proteins), mycocines, zymocines
and killer toxins (fungizidic proteins). The discovery II. Chromosomally Encoded
of antibiotics dates back to the beginning of the Killer Toxins
twentieth century and revolutionized medical anti-
infective therapies; however, the secretion of fungi- A. Williopsis
cidal proteins did not become obvious until the 1960s
A large variety of yeast species secrete chro-
1 mosomally encoded killer toxins. However, these
Institut für Molekulare Mikrobiologie und Biotechnologie,West-
fälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 appear to be exceptionally frequent among Will-
Münster, Germany; e-mail: meinhar@uni-muenster.de, roland. iopsis strains (Table 6.1). Among the most exten-
klassen@uni-muenster.de sively studied toxins are HM-1 produced by

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
Table 6.1. Chromosomally encoded yeast killer toxins
108

Species Strain Toxin Size Receptor Toxic activity References

Ascomycetes
Pichia anomala NCYC434 K5/panomycocin 49.0 b-1-3 Glucan Glucanase Izgü and Altinbay (2004)
WC65 83.3 b-1-6-Glucan Sawant et al. (1989)
ATCC 96603, K36, PaKT/PKT 85.0 b-Glucan (?) Guyard et al. (1999), Polonelli and Morace (1986)
UP25F
YF07b 47.0 Glucanase Wang et al. (2007a)
DBVPG 3003 Pikt >3.0 Comitini et al. (2004a)
P. farinosa KK1 SMKT a (6.6), Membrane permeabilisation Suzuki and Nikkuni (1994)
b (7.9)
P. kluyveri 1002 19.0 Membrane permeabilisation Middlebeek et al. (1979)
P. membranifaciens CYC1106 PMKT 18.0 b-1-6-Glucan Membrane permeabilisation Santos et al. (2000)
Williopsis californica DSM 12865 Wicaltin 34.0 Theisen et al. (2000)
W. saturnus IFO 0117 HYI 9.5 Inhibition of b-1,3-glucan Komiyama et al. (1995, 1998)
synthase
DBVPG 4561 KT4561 62.0 Buzzini et al. (2004)
W. saturnus var. mrakii MUCL 41968 WmKT 85.0 b-Glucan Glucanase/membrane Guyard et al. (2002a, 2002b)
permeabilization
IFO 0895 HM-1 10.7 b-Glucan Inhibition of b-1,3-glucan Ashida et al. (1983), Kasahara et al. (1994)
synthase
W. mrakii NCYC500 K500 1.8–5.0 b-Glucan Membrane permeabilisation Hodgson et al. (1995)
Debaryomyces hansenii CYC1021 23.0 b-1-6-Glucan Santos et al. (2002)
Kluyveromyces waltii IFO1666T >10.0 Kono and Himeno (1997)
K. wickerhamii DBVPG 6077 Kwkt >10.0 Comitini et al. (2004a)
K. phaffii DBVPG 6076 KpKt 33.0 b-1,6-/b-1,3- Glucanase Comitini et al. (2004b)
Glucan
K. marxianus NCYC587 K6 42.0 Izgü et al. (1999)
Friedhelm Meinhardt and Roland Klassen

Schwanniomyces ATCC 44252 a (7.4), Mannan Membrane permeabilisation Chen et al. (2000)
occidentalis b (4.9)
Saccharomyces 111 KHR 20.0 Goto et al. (1990)
cerevisiae
115 KHS 75.0 Goto et al. (1991)
Candida noadenis CnKT da Silva et al. (2008)
Basidiomycetes
Filobasidium IFM 40078 FC-1 b-1,6-Glucan Membrane permeabilisation Keszthelyi et al. (2006)
capsuligenum
Cryptococcus humicola VKM Y-1439 cellobiose lipid <1.0 Membrane permeabilisation Golubev and Shabalin (1994), Puchkov et al.
(2001, 2002)
 Yeast Killer Toxins: Fundamentals and Applications
W. saturnus var. mrakii IFO0895 (previously
known as Hansenula mrakii) and WmKT from
strain MUCL41968 of the same species (Ashida
et al. 1983; Kasahara et al. 1994; Guyard et al.
2002a).
HM-1 (known also as HMK) is a 10.7-kDa
toxin consisting of 88 amino acids (aa). Five in-
ternal disulfide bridges probably contribute to the
remarkable stability of the protein (Yamamoto
et al. 1986a, b). HM-1, the geneproduct of chro-
mosomal HMK, is produced as a precursor con-
sisting of 125 aa, that is post-translationally
processed upon secretion by the Kex2 endopepti-
dase (Fig 6.1; Kimura et al. 1993). HSK, an almost
identical toxin, is produced by W. saturnus
IFO0117 (Kimura et al. 1993). Such toxins exert
their effects on Saccharomyces cerevisiae by inhi-
biting the b-glucan synthase (Yamamoto et al.
1986; Komiyama et al. 1996), a plasma mem-
brane-bound enzyme catalysing the formation of
b-1,3-glucan, a major polysaccharide of yeast cell
walls (Lesage and Bussey 2006), eventually result-
ing in pore formation in actively growing cells in
regions in which cell wall synthesis is normally
active. As a consequence, intracellular material is
released and cells lyse concomitantly at budding
regions (Takasuka et al. 1995; Komiyama et al.
1996). Accordingly, osmotic stabilization in iso-
tonic medium reduced HM-1 toxicity (Komiyama
et al. 1996). In fact, cell wall glucan is a prerequisite
Fig. 6.1. Processing of toxin precursors. Toxin precursors
are schematically depicted on the left, mature proteins on
the right. Parts constituting mature toxins are given in
grey, parts being removed during processing are in
109
for HM-1 action since S. cerevisiae mutants dis-
playing a reduced glucan content (kre6) and spher-
oplasts are toxin-protected (Kasahara et al. 1994;
Komiyama et al. 2002).
Since HM-1 toxicity could be antagonized by
exogenously applied b-1-6- and b-1-3-glucan,
such polysaccharides were assumed to represent
the cell wall receptors (Kasahara et al. 1994). HM-1
has a basic isoelectric point (pI = 9.1) and is rather
unique in its broad pH and temperature stability,
as it retains its activity even after treatment at
100
C for 10 min and between pH 2 and pH 11
(Ashida et al. 1983; Lowes et al. 2000).
The three-dimensional structure of HM-1 was
elucidated by determining the nuclear magnetic
resonance spectrum (Antuch et al. 1996). The
toxin turned out to be a close structural homo-
logue of gB-crystallin, a monomeric protein origi-
nally isolated from bovine eye lenses.
Interestingly, HM-1 and gB-crystallin are not detectably
homologous at the aminoacid level (Antuch et al. 1996).
Other than gB crystallin and further eye lens crystallins,
which consist of two domains of the crystallin fold, HM-1 is
a single domain representative (Antuch et al. 1996). Pre-
sumably, eye lens crystallins and HM-1 have evolved from
an ancestral single domain precursor that underwent gene
duplication in the case of the crystallins. Resembling the
extreme thermal and pH characteristics of HM-1, crystal-
lins too exhibit outstanding stability features; they occur in
high concentration in eye lenses in which almost no protein
turnover takes place (Wistow and Piatigorsky 1988).
white. Subunit sizes are given in kDa; processing sites by
Kex1/Kex2 peptidases as well as signal peptidase (sp) are
indicated as such. –S–S– Disulfide bridge in mature toxins;
K1 contains multiple disulfide bridges
110 Friedhelm Meinhardt and Roland Klassen
The WmKT toxin of W. saturnus var. mrakii
strain MUCL41968 differs significantly from HM-
1 in structure and function; it is a 85-kDa protein
attacking a number of yeast species but, in con-
trast to HM-1, it has a relatively narrow pH opti-
mum and a low temperature optimum (i.e. around
pH 4.6 and 26–28
C; Guyard et al. 2002a), attri-
butes common to many other yeast killer toxins
(see below). Toxicity suppression by a glucosidase
inhibitor and in vitro glucosidase activity suggests
that WmKT kills target cells by degradation of cell
wall b-glucans (Guyard et al. 2002a). Consistently,
both spheroplasts and S. cerevisiae kre1 and kre4
mutants (the latter being defective in b-1-3- or b-
1-6-glucan synthesis) are protected from toxic
WmKT effects (Guyard et al. 2002a, b).
Other killer strains of the genus include W.
saturnus DBVPG 4561 and IFO 0117, W. califor-
nica DSM 12865 and W. mrakii NCYC500
(Komiyama et al. 1995; Hodgson et al. 1995; Thei-
sen et al. 2000; Buzzini et al. 2004; Table 6.1).
While the IFO 0117 toxin termed HYI is similar
to HM-1 in size, sequence and its inhibitory effect
on b-1-3-glucan synthase (Komiyama et al. 1995,
1998), the DBVPG4561 toxin is significantly larg-
er, with an estimated size of 62 kDa. It displays
heat and pH stability, as it is equally active at pH
values between 4.5 and 8.0 and tolerates up to
50 g/l NaCl. Elevated temperatures (30–45
C) de-
creased its activity only slightly; even treatments
of the toxin at 100
C – though administered rather
briefly – did not negatively influence its lethal
action (Buzzini et al. 2004). The exceptionally
small toxin of W. mrakii NCYC500 (1.8–5.0 kDa)
was found to be unstable at pH values above 4.0
and incubation temperatures exceeding 30
C
(Hodgson et al. 1995). Wicaltin, a toxin from W.
californica is a 34-kDa protein that displays a
broad spectrum of activity against pathogenic
and non-pathogenic yeast species (Theisen et al.
2000). As toxicity is partially suppressed in sorbi-
tol-stabilized yeast cells and the toxin dramatical-
ly affects protoplast regeneration (Theisen et al.
2000), wicaltin may inhibit b-1,3-glucan synthesis,
as for HM-1 of W. saturnus var. mrakii.
B. Pichia
Several strains of Pichia anomala secrete chro-
mosomally encoded toxins of varying molecular
masses (Table 6.1). Strain NCYC434 produces K5,
a 49-kDa toxin alternativley termed panomomy-
cin; it has exo-b-1,3-glucanase activity prefering
pH values between 3.0 and 5.5 and temperatures
up to 37
C (Izgü and Altinbay 2004; Izgü et al.
2005). Such enzymatic activity and the acidic pH
optimum reminds one of WmKT of W. saturnus
var. mrakii (Guyard et al. 2002a). Interestingly, a
monoclonal antibody raised against another
P. anomala killer toxin (strain ATCC 96603) dis-
played cross-reactivity with WmKT (Guyard et al.
2001), additionally supporting the assumption
that toxins of both taxa (Pichia, Williopsis) may
be structurally and functionally related, though it
still remains uncertain whether the toxin of P.
anomala ATCC 96603 is similar to the well char-
acterized K5 toxin (Guyard et al. 2001, 2002a, b;
Izgü and Altinbay 2005; Izgü et al. 2005). Since at
least the experimentally deduced molecular
masses of the toxins differ significantly (Table
6.1), both may only be distantly related.
Additional P. anomala killers include strains
WC65, K36, YF07b and DBVPG3003 (Table 6.1;
Sawant et al. 1989; Guyard et al. 1999; Comitini
et al. 2004; Wang et al. 2007a). As for K5, the
toxin of strain YF07b (isolated from marine en-
vironment for biocontrol of crab-pathogenic
yeast species in the food industry) displays b-
1,3-glucanase activity and has a similar molecu-
lar mass (47 kDa), raising the possibility that K5
and YF07b toxin are cognate proteins (Wang
et al. 2007a, b). Immunofluorescence analysis of
the 83.3-kDa toxin of strain WC65 revealed bind-
ing to both the cell wall and the membranes of
sensitive Candida albicans cells (Sawant et al.
1989; Sawant and Ahearn 1990). Since a S. cere-
visae kre1-1 mutant with reduced b-1-6 glucan
content displayed a significant degree of resis-
tance, b-1-6 glucan was suggested to be the pri-
mary cell wall receptor; possibly there is a
second, unidentified receptor in the plasma
membrane, which could explain the toxin-bind-
ing capability and sensitivity of spheroplasts
(Sawant and Ahearn 1990).
P. anomala strain DBVPG3003 was selected for
its ability to restrict growth of wine spoilage yeasts
(Comitini et al. 2004a). The toxin termed PiKt has
an acidic pH optimum (around pH 4.4) and is
inactivated above 40
C. However, it retained its
activity during prolonged incubation (10 days) in
a wine environment. The preliminary characteriza-
tion by ultrafiltration revealed a small molecular
mass toxin of 3–10 kDa (Comitini et al. 2004a).
 Yeast Killer Toxins: Fundamentals and Applications
P. kluyveri strain 1002 (Middelbeek et al. 1979)
produces a killer toxin which is stable between pH
2.5 and pH 4.7 and at temperatures below 40
C
(Middelbeek et al. 1979, 1980a). The toxin was
shown to form ion-permeable channels in phos-
pholipid bilayer membranes in vitro (Kagan 1983).
In vivo, cell death was accompanied by cell shrink-
age, leakage of potassium ions and adenosine 50 -
triphosphate, decrease of intracellular pH and
inhibition of the active uptake of amino acids
(Middelbeek et al. 1980a, b). Consistent with mem-
brane permeabilization as the lethal principle, tox-
icity could be suppressed when toxin treated cells
were plated on media containing physiological
concentrations of K+ and H+, whereas higher ion
concentrations had a clear negative effect on toxin
tolerance (Middlebeek et al. 1980b).
The two closely related halotolerant yeasts P.
membranifaciens and P. farinosa secrete toxins
termed P. membranifaciens killer toxin (PMKT)
and salt-mediated killer toxin (SMKT), respec-
tively, which are stimulated in the presence of
salt (Suzuki and Nikkuni 1989; Marquina et al.
1992; Lorente et al. 1997). SMKT, encoded by the
chromosomal SMK1 gene, is a preprotoxin of 222
aa (Suzuki and Nikkuni 1994). The precursor is
processed during secretion by the action of the
signal peptidase, as well as Kex1/2-like endo/car-
boxypeptidases, resulting in mature a- and b-
subunits of 6.6 kDa and 7.9 kDa, respectively;
simultaneously the interstitial g-polypeptide is
liberated (Fig. 6.1; Suzuki and Nikkuni 1994).
At pH values above 5.0, a- and b-subunits of
mature SMKT dissociate, resulting in the com-
plete loss of its activity (Suzuki et al 1997).
Determination of the crystal structure of SMKT revealed
that a and b subunits are folded together in a single
ellipsoidal domain (Kashiwagi et al. 1997). The primary
receptor of the toxin remains unknown, however, sensitiv-
ity of S. cerevisiae kre1 and kre5 mutants, being defective
in b-1-6 glucan synthesis suggest that molecules other
than b-1-6 glucan are required for target cell binding
(Suzuki and Nikkuni 1994).
Analysis of the interaction of SMKT with artificial
liposomes or intact yeast cells revealed that the
toxin disrupts membrane integrity (Suzuki et al.
2001).
The P. membranifaciens toxin PMKT is a
monomeric 18 kDa protein which kills sensitive
yeast cells such as Candida boidinii at tempera-
tures below 20
C and pH values below 4.8 (Santos
et al. 2000). Following binding to the primary
111
receptor b-1-6 glucan, the toxin is assumed to
form channels in the membrane, eventually caus-
ing leakage of ions and low molecular weight
metabolites such as glycerol (Santos et al. 2000;
Santos and Marquina 2004a). Thus, it resembles
the mode of action of P. kluyveri and S. cerevisiae
K1 toxins (see above and below). The characteri-
zation of the genome wide transcriptional re-
sponse of S. cerevisiae target cells to PMKT
exposure revealed the induction of genes of the
high glycerol (HOG) pathway (Santos et al. 2005),
which is implemented in the general environ-
mental stress response and which results in
increased levels of compatible solutes (such as
glycerol) to adapt the cellular osmotic pressure.
Hence, PMKT induces a co-ordinated transcrip-
tional response in target cells resembling the
response to osmotic stress (Santos et al. 2005;
Rep et al. 2000). Indeed, consistent with HOG
induction, intracellular and extracellular glycerol
accumulation were shown to be induced by
PMKT, as enhanced compatible solutes simulta-
neously leak out through PMKT-mediated mem-
brane channels (Santos et al. 2005). Strains
deficient in Hog1, the mitogen-activated protein
kinase (MAPK) integral to the HOG signalling
pathway as well as mutants defective in osmoa-
daptive glycerol synthesis display PMKT hyper-
sensitivity, clearly revealing compatible solute
accumulation to counteract PMKT toxicity
(Santos et al. 2005). Before reaching the plasma
membrane, PMKT was shown to interact with
Cwp2, a cell wall mannoprotein, the precursor
of which is attached to the plasma membrane
via a glycosylphosphatidylinositol (GPI) anchor,
while the mature Cwp2 localizes to the cell wall,
being covalently attached to b-1-6 glucan (Santos
et al. 2007). It was, thus, proposed that PMKT
first binds to the primary cell wall receptor b-1-6
glucan, followed by interaction with the mature
form of Cwp2, which is attached to b-1-6 glucan.
Subsequently, a third interaction with the GPI-
anchored form of Cwp2 facilitates close contact
with the plasma membrane, allowing lethal ion
channel formation (Fig. 6.2; Santos et al. 2007).
As for P. membranifaciens, a killer strain of Debaryomyces
hansenii (Table 6.1) isolated from high salt substrate (olive
brine) produces a toxin that is stimulated by high salt
concentrations and which also binds to b-1-6 glucan
(Lorente et al. 1997; Santos et al. 2002); however, the
mechanism of cell killing remains unknown.
112 Friedhelm Meinhardt and Roland Klassen
Fig. 6.2. Schematic representation of killer toxin mode of
action. b-1-6 glucan is the primary cell wall receptor for
chromosomally encoded PMKT and dsRNA encoded K1,
manno-protein serves as the receptor for dsRNA encoded
K28 and chitin, located close to the membrane is the
receptor for the dsDNA encoded zymocin. PMKT and K1
utilize GPI-anchored Kre1 and Cwp2 as secondary mem-
brane receptors and induce membrane channel formation.
C. Kluyveromyces
The genus Kluyveromyces harbours killer strains
secreting both, extranuclearly and chromosomally
encoded toxins (Tables 6.1, 6.2). While K. lactis
represents the most thougoughly investigated
plasmid encoded toxin (see below), chromosom-
ally encoded toxins are described for K. marxia-
nus, K. wickerhamii, K. waltii and K. phaffii
(Young and Yagiu 1978; Kono and Himeno 1997;
Izgü et al. 1999; Ciani and Fatichenti 2001; Comi-
tini et al. 2004a, b). The best characterized of the
latter, i.e. chromosomally encoded Kluyveromyces
toxins, is the 33-kDa KpKt of K. phaffii (Comitini
et al. 2004a). Based on competitive inhibition of
toxin activity, b-1-6/b-1,3 glucan was again iden-
tified as the cell wall receptor. The purified toxin
displayed glucanase activity, resembling the prop-
erties of W. saturnus var. mrakii WmKT and P.
anomala K5/panomomycin (Guyard et al. 2002a;
Comitini et al. 2004a; Izgü and Altinbay 2004; Izgü
et al. 2005). However, KpKt is much smaller than
WmKT or K5 and other than WmKT, it did not
induce rapid cell permeabilization as judged from
cell staining with propidium iodide, suggesting
differences in cell killing mediated by the two
glucanase toxins (Comitini et al. 2004a).
Cellular receptors or killing mechanisms for the
remaining chromosomally encoded Kluyveromyces
Erd2 was proposed as the secondary membrane receptor
for K28, which gains access to the target cell’s cytoplasm
by retrograde transport and finally inhibits DNA synthesis
in the nucleus. The secondary membrane receptor for
zymocin is unknown, however, Ipt1-synthesized sphingo-
lipid is required for cellular uptake of the toxin’s g-subunit
which cleaves cellular tRNAGlu. See text for references and
further details
toxins are currently unknown. Nonetheless, toxins
of K. phaffii, K. waltii and K. wickerhamii have been
investigated for their potential application for bio-
control during wine-making (see below).
For several other yeast species killer toxins are
known, a comprehensive compilation is given by
Golubev (2006); instances in which detailed infor-
mation about structural and mechanistic details
are available are presented in Table 6.1 Other
toxins have been isolated solely based on their
application potential, and basic information
about cell targeting and activity remains to be
established. In some cases, mycocinogenic activity
may resemble those of killer (protein-)toxins, but
in fact it is due to the release of toxic glycolipids
(Purchov et al. 2002, reviewed by Golubev 2006).
III. Extrachromosomally
Encoded Toxins
A. dsRNA Virus Toxins
Shortly after the discovery of the first yeast killer
in S. cerevisiae, it was realized that dsRNA viruses
constitute the molecular basis of toxin secretion
(Bevan et al. 1973). The S. cerevisiae killer viruses
belong to the totiviridae family. They persist as
a pair of two separately encapsulated virus like
 Yeast Killer Toxins: Fundamentals and Applications 113

Table 6.2. Extrachromosomally encoded yeast killer toxins

Species Strain Toxin Size Receptor Toxic activity References

Ascomycetes
dsRNA-encoded
S. cerevisiae KL88 K1 a (9.5), b-1-6-Glucan Membrane Young and Yagiu
b (9.0) permeabilisation (1978)
CBS8112 K28 a (10.0), Mannoprotein Blocking DNA Schmitt and Tipper
b (11.0) synthesis (1990)
Hanseniaspora uvarum 470 18.0 b-1-6-Glucan Schmitt and
Neuhausen
(1994)
Zygosaccharomyces 412 zygocin 10.0 Mannoprotein Membrane Schmitt and
bailii permeabilization Neuhausen
(1994)
dsDNA-encoded
Kluyveromyces lactis IFO1267 zymocin a (99.0), Chitin tRNAGlu-specific Gunge et al. (1981)
b (30.0), tRNase
g (28.0)
Pichia inositovora NRRL Y- >100.0 Chitin Hayman and Bolen
18709 (1991)
P. acaciae NRRL Y- PaT a (110.0), Chitin tRNAGln-specific Worsham and Bolen
18665 b (39.0), tRNase (1990)
g (38.0)
Debaryomyces CBS6693 >100.0 Chitin Klassen and
robertsiae Meinhardt
(2002)
Basidiomycetes
dsRNA-encoded
Ustilago maydis P1 KP1 13.4 Park et al. (1996a)
P4 KP4 13.6 Blocking calcium Park et al. (1994)
uptake
P6 KP6 a (8.6), K+ depletion Tao et al. (1990)
b (9.1)
Cryptococcus aquaticus VKM Y- Pfeiffer et al. (2004)
2428
Cystofilobasidium VKM Y- >15.0 Golubev et al. (2003)
infirmominiatum 2897
Trichosporon pullulans VKM Y- >15.0 Golubev et al. (2002)
2303
T. insectorum CBS10422 Fuentefria et al.
(2008)

particles in the host’s cytoplasm (for reviews, see
Wickner 1996, 1992; Schmitt and Breinig 2002,
2006). One of these is the L-A helper virus,
which encodes the major capsid protein (Gag)
and a RNA-dependent RNA polymerase.
The 50 end of the ORF encoding the RNA polymerase over-
laps in the –1 frame with the Gag-encoding ORF and is
expressed as a Gag-Pol fusion protein by a 1 ribosomal
frameshift event (Icho and Wickner 1989; Dinman et al.
1991). The L-A virus may be associated with different sate-
llite (non-autonomous) viruses, termed M-1, M-2 or M-28,
which encode a single preprotoxin that is processed during
secretion to yield the mature toxin. The capsid encoded by
L-A consists of 60 asymmetric Gag-dimers and two Gag-
Pol fusion proteins. The capsid is perforated allowing the
exit of (+)ssRNA transcribed from the viral genome within
the capsid as well as facilitating influx of host metabolites
but excluding entry of degradative enzymes or leakage of
dsRNA genomic molecules (Caston et al. 1997).
Replication of L-A and M viruses occurs in a
conservative manner (Fig. 6.3), with a (+)ssRNA
copy being generated in the capsid by the Gag-Pol
protein. Following export to the cytoplasm, such
ssRNA serves as the messenger that is translated
into preprotoxin (M-virus), or Gag and Gag-Pol
fusion proteins (L-A virus). The (+)ssRNA is sub-
sequently incorporated into newly formed cap-
sids, in which synthesis of the complementary
()RNA strand is carried out by Gag-Pol, closing
the replicative cycle.
114 Friedhelm Meinhardt and Roland Klassen
Fig. 6.3. Conservative replication cycle of the dsRNA toti-
viridae. Encapsulated virus like particles (VLP) are sche-
matically represented by a hexagon, they consist of Gag,
the major capsid protein and two Gag-RNA-polymerase
fusion proteins (Gag-Pol). +/ dsRNA double-stranded
RNA virus genome; + ssRNA single-stranded RNA tran-
scribed from the virus genome within the capsid by Gag-
Pol, which is encapsidated in new VLPs, followed by
synthesis of the complementary strand
L-A and M-viruses are considered virus like particles, since
they lack, in contrast to classic viruses, an extracellular
route of transmission. Propagation occurs exclusively by
cell to cell passage (anastomosis) during vegetative or
sexual reproduction. The most thoroughly studied viral
toxins from S. cerevisiae are K1 (encoded by M1 virus)
and K28 (encoded by M28 virus, see also Schmitt and
Breinig 2002, 2006).
1. K1
K1 is a dimer with subunit sizes of 9.5 kDa (a) and
9.0 kDa (b), which are covalently linked by three
disulfide bridges (Bostian et al. 1984). Both of the
subunits are formed by processing of a single
precursor protein termed preprotoxin, first by
the signal peptidase (giving rise to protoxin) and
subsequently by Kex1 (carboxypeptidase) and
Kex2 (endopeptidase) during secretion (Bostian
et al. 1984; Zhu et al. 1992). Kex2-processing
removes the g-peptide of the protoxin, a process
strikingly similar to the one occuring with the
chromosomally encoded SMKT (see above and
Fig. 6.1) and K28 (see below), despite the fact
that the toxins themselves do not display simila-
rities at the sequence level and also differ signifi-
cantly in their mode of action (de la Pena et al.
1981; Suzuki and Nikkuni 1994; Schmitt and Tip-
per 1995). K1 toxin, like many chromosomally
encoded toxins, first binds to b-1,6 glucan of the
cell wall (Hutchins and Bussey 1983). Subsequent-
ly, K1 interacts with Kre1, a GPI-anchored cell
wall protein, thereby inducing formation of (cat-
ion selective) ion channels within the plasma-
membrane (Fig. 6.2 de la Pena et al. 1978; Breinig
et al. 2002, 2004). Mutational analysis revealed
that both, a and b are involved in glucan binding,
while solely a is required for membrane interac-
tion, which agrees well with the presence of two
remarkably hydrophobic regions (Bussey 1991;
Zhu and Bussey 1991).
Whether ion channel formation is brought about by K1
alone (Martinac et al. 1990) or requires interaction with
primary membrane associated effector protein remains
controversial (Breinig et al. 2002; Breinig and Schmitt
2006). kre1 mutant spheroplasts were shown to be sensitive
to excess K1, raising the possibility of receptor-independent
membrane channel formation (Martinac et al. 1990) or,
alternatively, Kre1-independent access to a hypothetic pri-
mary membrane effector (Breinig et al. 2002). Although
evidence has been presented that the increase in membrane
permeability by K1 is due to the activation of Tok1 potassi-
um channels (Ahmed et al. 1999), full K1 sensitivity of tok1
cells indicates Tok1 activation to be (at best) a late second-
ary effect of the toxin (Breinig et al. 2002; Pagé et al. 2003).
Besides ionophoric action, K1 mode of action
strikingly resembles the chromosomally encoded
PMKT from Pichia membranifaciens, which has
also been shown to utilize a GPI-anchored cell wall
protein (Cwp2) as the secondary membrane re-
ceptor (Fig. 6.2 see above). As for Cwp2, Kre1 is
localized to both the cell membrane and cell wall,
as is typical for cell wall GPI-proteins which be-
come attached to glucan by transglycosylation
(Breinig et al. 2002). Thus, it appears possible
that both, K1 and PMKT use an identical strategy
for reaching the plasma membrane, involving se-
quential interaction with cell wall glucan, cell wall-
localized secondary receptors and, finally, mem-
brane-associated secondary receptor proteins.
As for PMKT, S. cerevisiae cells with a defect in
HOG signalling due to the loss of Hog1 display
extreme K1 sensitivity (Pagé et al. 2003), which
suggests a transcriptional response similar to
other osmotic stresses being effective in suppres-
sing K1 toxicity. However, while strains deficient in
intracellular glycerol accumulation, such as gpd1
(glycerol-3-phosphate dehydrogenase1) mutants
react hypersensitively to PMKT (Santos et al.
2005), this was not the case for K1 and no down-
stream effectors of Hog1 relevant for K1 resistance
could be identified (Pagé et al. 2003). Thus, while
 Yeast Killer Toxins: Fundamentals and Applications
the PMKT resistance promoting effect of HOG in-
duction involves osmo-adaptive compatible solute
production, its relevance for K1 resistance remains
to be elucidated (Pagé et al. 2003).
A characteristic trait of most extrachromoso-
mally encoded killer toxins is the appurtenant-spe-
cific toxin resistance (immunity), which is
genetically linked to toxin production (Schmitt
and Breinig 2006). In the case of K1, viral M1 RNA
displays a single ORF and the encoded preprotoxin
is not only processed to the mature secreted toxin
but also confers K1 immunity. Expression of a
cDNA copy of M1 in mutants which are disabled
in Kex2 function and therefore unable either to
secrete an active toxin or to carry out normal pro-
toxin processing and g-release confers K1 immuni-
ty. Further, expression of K1-a along with 31 N-
terminal residues of g is sufficient for K1 immunity
(Zhu et al. 1993). However, the exact immunity
strategy remains to be elucidated (Schmitt and Brei-
nig 2006) as the proposed mechanism based on
internal inhibition of Tok1 potassium channels by
K1 precursors is questionable (see above).
Another S. cerevisiae dsRNA virus termed M2
encodes the K2 toxin, which is assumed to exhibit a
rather similar toxic activity and apparently also
uses Kre1 as the plasma membrane receptor; how-
ever, the primary K2 sequence is unrelated to K1,
and K2 displays a slightly more acidic pH optimum
(Young and Yagui 1978; Pfeiffer and Radler 1984;
Dignard et al. 1991; Schmitt and Breinig 2002;
Novotna et al. 2004). As K2 killers remain suscepti-
ble to K1 (and vice versa) and differential resis-
tance effects of a kre2 mutation to both toxins were
observed, functional difference with respect to tar-
get cell interactions and immunity mechanisms are
obvious, the details of which, however, remain to
be uncovered (Dignard et al. 1991; Meskauskas and
Citavicius 1992; Novotna et al. 2004).
2. K28
The other well characterized RNA viral toxin in
Saccharomyces is K28, encoded by the M28 virus.
Very similar to K1, K28 is encoded by a single
ORF as a preprotoxin, which is processed during
secretion by the signal peptidase as well as Kex1
and Kex2 peptidases (Schmitt and Tipper 1990,
1995; Riffer et al. 2002; Schmitt and Breinig 2006).
As for K1, the a-b intervening g-peptide of K28
protoxin is deleted by the Kex2 endopeptidase.
Mature K28 consists of two subunits (a, b) of 10
115
kDa and 11 kDa, which are covalently linked by a
single disulfide bridge (Schmitt and Tipper 1995).
Despite this striking similarity, subsequent steps
in target cell attack are completely different for K1
and K28. In fact, the latter is so far the only
instance of a yeast killer toxin gaining access to
the target cell by endocytosis and subsequent ret-
rograde passage of the secretory pathway, fol-
lowed by exit from the endoplasmic reticulum
(ER) and entry into the nucleus, where it blocks
DNA replication (Fig. 6.2 Schmitt et al. 1996; Eis-
feld et al. 2000; Heiligenstein et al. 2006).
K28 first binds to mannoprotein in the cell wall, most
likely followed by interaction with Erd2 which functions
as membrane receptor (Schmitt and Radler 1987; Schmitt
and Breinig 2006). Erd2 is an integral membrane protein
that binds to the HDEL motif in proteins destined for
retention in the endoplasmic reticulum (Semenza et al.
1990) and it is assumed that a low number of Erd2 also
reach the cytoplasmic membrane where they may contact
the K28 b-subunit (Schmitt and Breinig 2006). Kex1-pro-
cessing of K28 b at the C-terminal HDELR motif uncovers
the ER retention signal, which is essential for re-entry into
the secretory pathway of the target cell. Consistently, K28
derivatives lacking the HDELR motif are inactive and cells
defective in Erd2 no longer internalize the toxin (Eisfeld
et al. 2000). Endocytotic uptake probably involves (mono-
)ubiquitination of the membrane-receptor by Uba1, Udc4
and Rsc5 and, subsequently, retrograde passage of K28
from endosomes to the ER occurs, from where it exits to
the cytoplasm via the Sec61 complex (Eisfeld et al. 2000;
Heiligenstein et al. 2006). The Sec61 translocon is a bidi-
rectional membrane channel which mediates translocation
of secretory proteins into the ER but also removes mis-
folded proteins from the secretory pathway for subsequent
degradation by the ER-associated protein degradation
(ERAD) pathway (reviewed by Nakatsukasa and Brodsky
2008). K28 apparently masquerades as an ERAD substrate
and the ER chaperones Kar2, Pdi1, Scj1, Jem1 and Pmr1
assist the exit of partially unfolded a-b subunits from the
ER. Once the cytosol is reached, b is ubiquitinated and
degraded by the proteasome, while a enters the nucleus
and inhibits DNA synthesis (Schmitt et al. 1996; Heiligen-
stein et al. 2006).
As for K1, K28 preprotoxin not only confers the
killer phenotype to M28-infected cells but also
renders toxin producers immune against their
own toxin. Moreover, K28 immunity is also estab-
lished in a kex2 mutant, which is unable to release
active toxin, as described above for K1 (Schmitt
and Tipper 1992; Zhu et al. 1992). While the
mechanism of K1 immunity is still unknown,
details for K28 were recently uncovered (Breinig
et al. 2006). Immune K28 killer cells reinternalize
their own toxin and subsequent interaction of the
116 Friedhelm Meinhardt and Roland Klassen
latter with unprocessed preprotoxin in the cyto-
plasm is the key step for immunity, which
involves ubiquitination and selective proteasomal
degradation of mature K28 (Breinig et al. 2006).
Interestingly, partial immunity could also be
established when only the a-subunit of the toxin
is expressed in the cytoplasm and full immunity
requires only a non-specific sequence extension to
the a-subunit, which strikingly resembles the sit-
uation in K1 immunity (Breinig et al. 2006).
3. Other dsRNA Virus Toxins
Shortly after the discovery of the killer phenotype
in S. cerevisiae an extranuclear inherited killer phe-
nomenon was recognized in the dimorphic fungus
Ustilago maydis (Puhalla 1968), a basidiomycete
which can grow like a budding yeast or switch to
filamentous growth (reviewed by Steinberg and
Perez-Martin 2007). Three different immunity spe-
cificities (P1, P4, P6) are reported to exist in natural
killer isolates, all of which are encoded by dsRNA
viruses (Koltin and Day 1976; Tipper and Bostian
1984). While KP1 and KP4 are monomeric proteins
of 13.4 kDa and 13.6 kDa, respectively (Park et al.
1994, 1996a), KP6 is a dimer consisting of subunits
of 8.6 kDa and 9.1 kDa (Tao et al. 1990). Structure
determination of KP4 revealed that the toxin
belongs to the a/b-sandwich fold, displaying weak
similarities to scorpion toxins, which exert their
neurological effects by acting on Na+ channels
(Gu et al. 1995). Rescuing KP4 treated sensitive U.
maydis cells by supplying exogenous Ca2+ sug-
gested toxin action by inhibition of Ca2+ channels
(Gu et al. 1995). Such a hypothesis was further
substantiated by demonstrating KP4-mediated in-
hibition of voltage-gated Ca2+ channels in several
types of mammalian neuronal cells (Gu et al. 1995).
Crystallization of SMKT from Pichia farinosa (see
above) surprisingly revealed close structural simi-
larities of KP4 and SMKT (Kashiwagi et al. 1997),
despite the fact that the latter is a chromosomally
encoded dimeric protein, assumed to interact and
disrupt cytoplasmic membranes of target cells
(Suzuki et al. 2001).
In contrast, KP6 is composed of two subunits
as for the S. cerevisiae dsRNA encoded toxins;
however, the two subunits are not covalently linked
by disulfide bonds (Peery et al. 1987; Tao et al.
1990). The toxic effect, which probably involves
K+ depletion, requires both of the subunits but,
interestingly, they do not directly interact in solu-
tion (Peery et al. 1987). Crystallization of the smal-
ler KP6 (a)-subunit revealed a third killer toxin
member of the a/b sandwich fold; however, other
than SMKT and KP4, KP6a subunits form a hex-
americ assembly with a central pore which may
provide a structure of the appropriate dimension
to generate a membrane K+ channel (Li et al. 1999).
Basidiomyceteous yeasts belonging to the
genera Trichosporon, Cryptococcus and Cystofili-
basidium harbour virus-like particles with dsRNA
genomes, which are associated with killer (alter-
natively termed mycocinogenic) phenotypes
(Golubev et al. 2002, 2003; Pfeiffer et al. 2004;
Fuentefria et al. 2008). Though basic characteris-
tics of the toxins, including spectra of target yeasts
and pH or temperature optima were investigated,
it remains to be elucidated whether their struc-
tures or mode of action resemble known dsRNA
or chromosomally encoded toxins.
The ascomycetous yeasts Hanseniaspora
uvarum and Zygosaccharomyces bailii also contain
virus-like particles with toxin-encoding dsRNA
genomes (Zorg et al. 1988; Radler et al. 1990).
Similar to S. cerevisiae, both of the former contain
autonomous L- and accompanying toxin-encoding
M-viruses (Schmitt and Neuhausen 1994; Schmitt
et al. 1997; Weiler et al. 2002). The H. uvarum toxin
is a 18-kDa monomer, which uses b-1-6 glucan as
the cell wall receptor; it displays a broader killing
spectrum than S. cerevisiae killers (Radler et al.
1990; Schmitt and Neuhausen 1994; Schmitt et al.
1997). The Z. bailii toxin (termed zygocin) likewise
exhibits a broad action spectrum, including human
and phytopathogenic fungi (Weiler and Schmitt
2003). The 10-kDa zygocin rapidly induces per-
meabilization, probably due to membrane channel
formation (Weiler and Schmitt 2003; Schmitt and
Breinig 2006). In contrast to the other dsRNA-
encoded toxins, zygocin immunity is not mediated
by the preprotoxin precursor. Since intact cells and
protoplasts of Z. bailii are naturally resistant to
zygocin, specific immunity is obviously dispens-
able, a situation that is also realized for chro-
mosomally encoded toxins (Weiler et al. 2003).
B. Linear Plasmid-Encoded Toxins
Linear dsDNA plasmids which persist in the
cytoplasm are related to viruses with respect to
gene content and mode of replication; they were
 Yeast Killer Toxins: Fundamentals and Applications
originally detected in a killer strain of Kluyvero-
myces lactis (Gunge et al. 1981). Today, such cyto-
plasmic DNA elements are known to occur in
several ascomycetous yeasts belonging to different
genera (such as Pichia, Candida, Debaryomyces,
Saccharomycopsis, Schwanniomyces, Botryoascus)
but also in the basidiomycete Trichosporon pull-
ulans (Kitada and Hishinuma 1987; Ligon et al.
1989; Worsham and Bolen 1990; Hayman and
Bolen 1991; Bolen et al. 1992; Cong et al. 1994;
Fukuhara 1995; Chen et al. 2000).
In addition to K. lactis, three other species
(P. acaciae, P. inositovora, D. robertsiae; Table 6.2)
contain a set of two or three linear plasmids confer-
ring a killer phenotype (Fig. 6.4; Worsham and
Bolen 1990; Hayman and Bolen 1991; Klassen and
Meinhardt 2002). Though such dsDNA molecules
are probably not encapsulated, a general functional
partition among the plasmids of each species resem-
bles the L- and M-viruses of Saccharomyces cerevi-
siae. Invariably, there is a highly conserved
autonomous element, which provides factors
essential for cytoplasmic gene expression and repli-
cation (reviewed by Jeske et al. 2006a; Klassen
and Meinhardt 2007). Such element can exist solely
or together with a non-autonomous element encod-
ing a killer toxin. As expression of toxin genes and
replication of the element requires functions
provided by the autonomous element, it reminds
one of the L-dependency of M-viruses (see above).
Replication of both autonomous and non-autonomous
elements is initiated by protein priming, involving a viral
B-type DNA polymerase fused to the terminal protein that
remains covalently bound to the 50 ends of the plasmids
Fig. 6.4. Schematic representation of virus like dsDNA
plasmids from yeasts encoding killer toxins. Based on
sequence similarity and gene content, two subgroups are
defined. Arrows indicate ORFs and their transcriptional
direction; terminal proteins are depicted as filled circles;
terminal inverted repeats correspond to filled triangles.
117
(Hishinuma et al. 1984; Stark et al. 1984; Sor and Fukuhara
1985; Tommasino et al. 1988; Hishinuma and Hirai 1991;
Klassen et al. 2001; Klassen and Meinhardt 2003; Jeske and
Meinhardt 2006).
Cytoplasmic transcription of linear plasmid-encoded
genes involves a rather unique plasmid-encoded RNA
polymerase and a mRNA-capping enzyme which resem-
bles the capping enzyme of the cytoplasmic vaccinia virus
(Wilson and Meacock 1988; Larsen et al. 1998; Tiggemann
et al. 2001; Klassen and Meinhardt 2007). The plasmid-
encoded RNA polymerase recognizes unique cytoplasmic
promoters with a short consensus sequence (6 nt) which is
incompatible with the nuclear transcription machinery
(Romanos and Boyd 1988; Kämper et al. 1989a, b; Kämper
et al. 1991; Schickel et al. 1996).
The four known toxin-encoding killer plasmids
consist of two subgroups judged from overall
similarity, gene content and genetic prerequisites
for toxin function (Fig. 6.4 see below). Group I
comprises the K. lactis and P. inositovora killer
plasmids while group II harbours the P. acaciae
and D. robertsiae killers (Schaffrath and Mein-
hardt 2004; Jeske et al. 2006b; Klassen and Mein-
hardt 2007).
1. Group I
The K. lactis killer system consists of the autono-
mous element pGKL2 and the toxin (zymocin)-
encoding non-autonomous pGKL1 (for a review,
see Stark et al. 1990). Zymocin is a heterotrimeric
(a, b, g) glycoprotein, the subunits of which dis-
play molecular masses of 99, 30 and 28 kDa, re-
spectively (Stark and Boyd 1986). While the
smallest subunit (g) is encoded separately, the
Known or proposed functions of encoded proteins are
indicated. toxin Intracellular toxic subunit, imm immunity
proteins. Similarity at the sequence level for either toxic
subunits or immunity proteins is indicated by grey
shading. The predicted toxin uptake protein is homolo-
gous in all systems
118 Friedhelm Meinhardt and Roland Klassen
two larger ones are formed from a single genepro-
duct (Orf2) by endopeptidase processing during
secretion (involving the S. cerevisiae Kex2 homo-
logue of K. lactis, Kex1; see Fig. 6.1 Hishinuma
et al. 1984; Stark et al. 1984; Sor and Fukuhara
1985; Stark and Boyd 1986; Tokunaga et al. 1987;
Stark et al. 1990). In the mature zymocin, b and g
are covalently linked via a disulfide bond and the
a-subunit contains at least one internal disulfide
bridge (Stark and Boyd 1986; Stark et al. 1990).
Zymocin differs from many other yeast killer toxins in
having a neutral pH optimum and additionally exhibits
relatively high thermal stability (no loss of activity fol-
lowing incubation at 50
C; Butler et al. 1991a). Cell
killing by zymocin involves cell wall binding of the
complex by virtue of its largest (a)-subunit, which has
a cysteine-rich chitin binding and a LysM domain, the
latter also found in peptidoglycan binding proteins
(Stark et al. 1990; Butler et al. 1991b; Jablonowski et al.
2001; Jeske et al. 2006a). The a-subunit further exhibits
clear similarities to family 18 chitinases and displays
exochitinase activity which is apparently essential for
toxicity (Butler et al. 1991b; Jablonowski et al. 2001).
Cell-killing activity, however, resides in the smallest sub-
unit of zymocin, as conditional intracellular expression
of solely this subunit is toxic and also results in a G1 cell
cycle arrest induced by exo-zymocin (Tokunaga et al.
1989; White et al. 1989; Stark et al. 1990; Butler et al.
1991c; Lu et al. 2005). Cellular uptake of the g-subunit
occurs in an unknown manner, probably with participa-
tion of the a-subunit’s chitin binding and chitinase ac-
tivity and the remarkably hydrophobic b-subunit
(Fig. 6.2). Also involved is the plasmamembrane sphin-
golipid mannosyl-diinositolphospho-ceramide M(IP)2C
(synthesized by the Kti6/Ipt1 protein) and the plasma
membrane ATPase Pma1; both are conjointly required
for toxin action from outside (Mehlgarten and Schaffrath
2004; Zink et al. 2005). In contrast to chitin-deficient
(chs3) cells, which lack zymocin binding, an ipt1 mutant
is proficient for binding but denies g-import (Zink et al.
2005), revealing the M(IP)2C-involving step to occur
subsequent to initial toxin recruitment to the cell wall.
Whether M(IP)2C has a function similar to the secondary
membrane receptor for other toxins (see above), remains
unknown at present.
Following membrane passage, g apparently exists
in a transiently dormant form which requires in-
tracellular activation by the plasma membrane
H+ATPase Pma1 or the membrane potential gen-
erated by this enzyme (Mehlgarten and Schaffrath
2004; Zink et al. 2005). In the absence of function-
al Pma1, g-activation could be induced experi-
mentally by acidification of the cell exterior,
though it remains unclear which process is affect-
ed by H+ flux (Mehlgarten and Schaffrath 2004).
Once inside the cell, g specifically cleaves
tRNAGlu within the anticodon loop, at the 30 side
of the wobble uridine (U34; Lu et al. 2005). A post-
transcriptional modification, which is present at the
uracil-C5 position of U34 in tRNAGluUUG, a 5-meth-
oxy-carbonyl-methyl (mcm5) residue, is crucial for
cleavage (Lu et al. 2005; Jablonowski et al. 2006).
mcm5 synthesis in tRNAGlu and several other tRNAs with
an uridine residue at the wobble position requires the six-
subunit Elongator complex (Elp1–6) as well as the tRNA
methyltransferase Trm9 which is assumed to function
downstream of Elongator in the final step of mcm5 synthe-
sis (Kalhor and Clarke 2003; Huang et al. 2005; Lu et al.
2005). Consistent with the importance of mcm5 for cell
killing by the tRNase, loss of ELP1-6 or TRM9 prevents
zymocin toxicity (Frohloff et al. 2001; Lu et al. 2005; Jablo-
nowski et al. 2006) and complementation of spontaneous
killer toxin insensitive (kti) S. cerevisiae mutants identified
further loci with a previously unknown role in tRNA mcm5
modification (Fichtner and Schaffrath 2002; Fichtner et al.
2002; Huang et al. 2005; Lu et al. 2005; Jablonowski and
Schaffrath 2007; Zabel et al. 2008).
In vitro analysis revealed additional cleavage of two
other mcm5-modified tRNAs, i.e. tRNAGln and
tRNALys, by the heterologously expressed toxic
subunit of zymocin, although the efficiency was
much lower when compared to tRNAGln (Lu et al.
2005). Contrary to the latter, no depletion of
tRNAGln or Lys was seen in cells treated with zymo-
cin. However, as combined overexpression of
tRNAGlu, Gln and Lys intensified zymocin resistance
when compared to a strain overexpressing tRNAGlu
solely, the glutamine and lysine tRNAs were as-
sumed to represent zymocin targets in vivo, in
addition to tRNAGlu (Lu et al. 2005).
A killer strain of Pichia inositovora contains a
set of three plasmids (pPin1-1, pPin1-2, pPin1-3)
of which pPin1-2 is dispensable for killer activity
(Hayman and Bolen 1991); As for K. lactis, the
larger pPin1-1 probably represents the autono-
mous element whereas the toxin is encoded by
the smaller pPin1-3 (Klassen and Meinhardt
2003). The P. inositovora toxin displays a molecu-
lar mass larger than 100 kDa and probably con-
sists of at least two subunits (Klassen and
Meinhardt 2003). The largest subunit (100
kDa) is similar to zymocin a- and b and – as for
zymocin – was proven to bind chitin in vitro.
Disruption of the chitin synthase III-encoding
gene in a sensitive S. cerevisiae strain resulted in
insensitivity to the P. insitovora toxin as well.
As the pPin1-3 killer plasmid also encodes a
 Yeast Killer Toxins: Fundamentals and Applications
homologue of the zymocin’s tRNase subunit and
toxicity requires the Elongator complex involved
in tRNA-mcm5 modification, a similar mode of
action of the P. inositovora toxin and zymocin is
likely to occur (Klassen and Meinhardt 2003).
Despite such striking similarities, zymocin
and the P. inositovora toxin clearly differ with
respect to target cell spectra and pH optima; in
contrast to the broad spectrum of activity with a
neutral pH optimum of zymocin, P. inositovora
toxin has an acidic pH optimum and a clearly
narrower spectrum of target yeasts (Hayman and
Bolen 1991). Sensitive reaction to P. inositovora
toxin is apparently not species-specific, as two
related S. cerevisiae strains are killed by the
toxin, while several other laboratory strains sensi-
tive to zymocin display resistance to P. inositovora
toxin (Hayman and Bolen 1991; Klassen and
Meinhardt, unpublished data).
The zymocin-encoding plasmid (pGKL1)
harbours (in addition to the toxin-encoding
ORF) a gene responsible for the specific related
immunity, the mechanism of which, however,
remains to be elucidated (Tokunaga et al. 1987).
Despite the fact that a gene homologous to the
zymocin immunity factor is located on the P.
inositovora killer plasmid pPin1-3 (Klassen and
Meinhardt 2003), the latter is not required for
immunity in this species (Hayman and Bolen
1991). Possibly, intrinsic immunity has evolved
subsequent to the acquisition of the killer plas-
mid system, which concomitantly made a plas-
mid-encoded immunity function redundant. In
K. lactis, the plasmid-encoded immunity func-
tion establishes an auto-selection system for the
entire plasmid set. Spontaneous loss of the non-
autonomous element alone or together with the
autonomous element would give rise to zymocin
() and sensitive cells which are eliminated due
to the stable zymocin toxin in the environment.
A very similar autoselection system is also pres-
ent in most dsRNA- and other dsDNA-encoded
killer systems (see above and below). It cannot be
decided whether toxin/immunity functions are
recruited by viral or virus-like genomes persist-
ing in the yeast cell’s cytoplasm for the purpose
of creating a selective advantage for the host
(elimination of competitors) or stable propaga-
tion of the virus or both. However, in cases where
intrinsic immunity is established, a toxin-depen-
dent autoselection effect on the respective ele-
ment is no longer present.
119
2. Group II
The two killer plasmids of homology group II,
pPac1-2 from P. acaciae and pWR1A from D.
robertsiae are similar with respect to genome or-
ganization (Klassen et al. 2004). pWR1A harbours
one additional gene, which is not present in
pPac1-2 (Fig. 6.4). The pPac1-2 encoded toxin
(PaT) was purified and found to consist of three
subunits with molecular masses of 110, 39 and 38
kDa, respectively (McCracken et al. 1994). The
pWR1A-encoded toxin too is a larger protein of
above 100 kDa and both, PaT and D. robertsiae
toxin display neutral pH optima (McCracken et al.
1994; Klassen and Meinhardt 2002). The similarity
of the toxin-encoding plasmids to homology
group I elements (pGKL1, pPin1-3) is restricted
to the ORF encoding the chitin-binding polypep-
tide (a-subunit), whereas separately encoded and
potentially secreted proteins (pPac1-2 Orf2p,
pWR1A Orf3p) are not related to zymocin g and/
or the P. inositovora equivalent (Klassen et al.
2004). However, as intracellular expression of
these genes mimics the effects of extracellularly
applied toxins on target cells, the toxic activity
resides in such separately encoded toxin subunits,
which act intracellularly on target cells, as for
zymocin g (Klassen et al. 2004). Other than zymo-
cin, neither of the toxins encoded by group II
plasmids requires the Elongator complex for func-
tion; however, PaT was recently shown to target
cellular tRNA, as for zymocin (Klassen et al. 2008).
The tRNase activity of PaT is specific for tRNAGln
(in contrast to zymocin, which attacks tRNAGlu)
and loss of mcm5 modification does not prevent
substrate cleavage in vivo or in vitro. Interesting-
ly, however, an mcm5 biosynthesis intermediate
(carboxyl-methyl-residue; cm5), which presum-
ably exists in the tRNAGln of cells deficient in the
tRNA methyltransferase Trm9, strongly inhibits
cleavage and, thus, largely prevents PaT toxicity.
In vitro analysis of cleavage products revealed two
alternative cleavage sites in tRNAGln, one of which
is the mcm5 modified wobble nucleoside U34 and
the other is most probably U32. Loss of mcm5
prevented cleavage at position U34, but not at
U32, explaining the sensitive phenotype of Elon-
gator mutants to PaT. The negative charge of cm5
might be the reason for its inhibitory effect on
cleavage at both positions (Klassen et al. 2008).
As for zymocin, the heterologously expressed
toxic subunit of PaT displayed an extended
120 Friedhelm Meinhardt and Roland Klassen
substrate spectrum, not only accepting tRNAGln
but with reduced efficiency also mcm5-modified
tRNAGlu and Lys (Klassen at al. 2008). Cleavage of
all these substrates was (at least partially) inhib-
ited by cm5, but not by the entire loss of the mcm5
modification of U34, which is in marked contrast
to zymocin’s tRNase activity and may be
explained by the existence of an alternative cleav-
age site (see above).
Interestingly, cell killing by both zymocin and
PaT involves the occurrence of DNA damage, pre-
sumably as a downstream consequence of tRNA
depletion (Klassen et al. 2007, 2008). Though not
systematically analysed yet, it is speculated that
tRNase toxin exposure may lead to deficiency in
translation of messengers relevant for genome
surveillance during replication. Consistently, ge-
netic analysis revealed strong evidence for the
occurrence of replication-derived DNA double-
strand breaks in both PaT- and zymocin-exposed
cells and DSB repair by homologous recombina-
tion was found to confer resistance against both
toxins. An alternative DSB repair pathway (non-
homologous end-joining), which differs from ho-
mologous recombination in its inability to operate
on replication-derived DSBs, is even disadvanta-
geous for PaT/zymocin survival and this effect is
due to inhibition of homologous recombination
(Klassen et al. 2006, 2007, 2008). Clearly, however,
further work is required to understand the cellular
consequences of tRNA attack by specific endonu-
cleases in yeast, as such a toxic principle was only
quite recently recognized in eukaryotic toxins (Lu
et al. 2005; Klassen et al. 2008). Interestingly, PaT
and zymocin both do not deplete their target
tRNAs completely and, though both significantly
impair translation, there is no complete shut-off
of protein biosynthesis (Butler et al. 1991a; Klas-
sen and Meinhardt, unpublished data). Moreover,
PaT was shown to induce apoptotic cell death
under conditions where only a fraction of target
cells is rapidly killed; the remaining initially sur-
viving fraction later executed an active cell death
program involving protein biosynthesis and the
classic apoptotic hallmarks, including nuclear
fragmentation, ROS production and phosphati-
dyl-serine externalization (Klassen and Meinhardt
2005). As other toxins, such as K1, K28 and zygo-
cin also induce apoptotic cell death when applied
in moderate doses (Reiter et al. 2005); such cellu-
lar response is apparently not functionally
connected to the primary lethal principle of the
individual toxins, but rather a common reaction
of individual cells to life-threatening conditions.
With the exception of pPin1-3, all killer plas-
mids carry an additional gene which is responsi-
ble for a specific immunity phenotype (Tokunaga
et al. 1987; Paluszynski et al. 2007; Paluszynski
and Meinhardt, unpublished data). Though the
mode of action of the encoded immunity proteins
remains to be elucidated, they disable the effects
of the respective toxic subunit at the intracellular
level, as for the only other toxin known to act
intracellularly on target cells, the M28-encoded
K28 toxin (Tokunaga et al. 1989; Breinig et al.
2006; Paluszynski et al. 2007). Thus, in all known
killer/immunity systems where toxicity requires
cellular uptake of the toxin (zymocin, PaT, K28),
toxin uptake apparently also occurs in the toxin
producer and the reinternalized toxin is specifi-
cally degraded by the host cell’s proteasome (K28)
or disposed by yet unknown mechanisms (zymo-
cin, PaT).
IV. Applications
A. Antifungals for Human Therapy
Several yeast killer toxins, such as W. mrakii var.
mrakii HM-1, W. saturnus toxin, P. anomala
PaKT and Zygosaccharoymces bailii zygocin,
exhibit a broad spectrum of activity including
human pathogenic yeasts, such as Candida albi-
cans and Cryptococcus neoformans. Consequently,
they are suggested to be potentially useful for
therapy of human infections caused by fungal
pathogens (Yamamoto et al. 1988; Walker et al.
1995; Magliani et al. 1997a; Theisen et al. 2000;
Weiler and Schmitt 2002; Buzzini et al. 2004; Izgü
et al. 2007a). However, direct application of killer
toxins is of limited practical importance because
many of these proteins are unstable or inactive at
temperatures around 37
C or neutral pH. Further-
more, antigenicity and toxicity may prohibit ap-
plication in the human bloodstream (Magliani
et al. 2004). Yet, some killer toxins (e.g. the Will-
iopsis toxins) display a remarkable temperature
stability, which might facilitate their use as topical
applications on superficial skin lesions (Buzzini
et al. 2004). P. anomala K5 toxin exhibiting ac-
ceptable temperature stability was studied against
dermatophytes and several pathogenic Candida
species. All clinical isolates as well as type strains
 Yeast Killer Toxins: Fundamentals and Applications
belonging to the genera Trichosporon, Micro-
sporum and Candida were found to be susceptible
to K5, suggesting this toxin to be applicable as a
topical antifungal agent (Izgü et al. 2007a, b).
A rather promising approach to overcome the
above-mentioned problems associated with the
direct application of killer toxins was initiated by
Polonelli and Morace (1988). A monoclonal anti-
body (mAbKT4), which neutralized the in vitro
activity of P. anomala UCSC 25F (= ATCC 96603;
Table 6.1) PaKT (Polonelli and Morace 1987) was
used to raise anti-idiotypic antibodies displaying
an internal image of the toxin’s active side. Strik-
ingly, such natural polyclonal, as well as subse-
quently developed monoclonal antibodies or
single-chain variable fragments (scFv) derived
from a phage display library were able to interact
with the cell wall and kill yeast cells susceptible to
the original P. anomala toxin (Polonelli and Mor-
ace 1988; Polonelli et al. 1990, 1997; Magliani et al.
1997a; Magliani et al. 2004). Vaccination with
mAbKT4 in the mouse model resulted in the pro-
duction of killer toxin-like antibodies, which con-
ferred significant protection against experimental
candidiasis (Polonelli et al. 1993, 1994).
A completely novel approach for the treat-
ment of vaginal candidiasis was realized with the
construction of genetically engineered Streptococ-
cus gordonii strains, being able to colonize vaginal
mucosal sites and either displaying killer toxin-
like antibodies (ScFv) at the cell surface or secret-
ing them, resulting in anti-candida activity of po-
tential therapeutic value (Magliani et al. 1997b).
Decapeptides derived from scFv and displaying
P. anomala toxin-like activity (so-called mimo-
topes) were subsequently identified and further
optimized by alanine scanning, resulting in the
extremely stable fungizidal killer peptide KP
(Polonelli et al. 2003), which is active against a
broad variety of pathogenic micro-organisms, in-
cluding Cryptococcus neoformans and Paracocci-
dioides brasiliensis (Cenci et al. 2004; Travassos
et al. 2004). Killer toxin-like antibodies or killer
peptides derived from PaKT were shown to addi-
tionally kill a variety of pathogenic prokaryotic
micro-organisms, such as Mycobacteria, Staphy-
lococcus, Streptococcus species and plant-patho-
genic Pseudomonas strains (reviewed by
Magliani et al. 2004). It was proposed that b-glu-
can serves as the yeast cell wall receptor for P.
anomala toxin, as for the related WmKT from
W. saturnus var. mrakii (see also above) and
121
thus, glucan or glucan-like molecules in the cell
wall of susceptible pro- and eukaryotic micro-
ogranisms were proposed to provide the basis
for the observed extremely broad spectrum of
toxin activity, idiotypic antibodies and derived
decapeptides (Magliani et al. 2004).
In addition to P. anomala toxin, HM-1 from
W. saturnus var. mrakii was used to produce toxin
neutralizing antibodies that were subsequently
employed in idiotypic vaccination and the pro-
duction of killer toxin-like antibodies, which
display an internal image of HM-1’s active site
and inhibit the target cell’s glucan synthase
activity (Selvakumar et al. 2006a, b, c). As for the
P. anomala toxin, such killer activity-bearing
antibodies may have application potential in
the treatment of human fungal infections and
may possibly also be developed to recombinant
antimycotic-producing bacteria or mimotopes
displaying the desired activity. Small peptide
mimotopes can potentially be produced much
more economically when compared to immuno-
logical killer toxin derivatives. Consequently, the
P. anomala toxin-derived KP was patented and
has entered clinical trials, possibly ending up as
the first member of a new generation of antifungal
agents derived from yeast killer toxins (Magliani
et al. 2004).
B. Antifungals in Agriculture, Food
and Feed Industry
There is a clear application potential for yeast
killer toxins in the wine industry. The wine envi-
ronment is characterized by low pH (3.5), at
which some of the chromosomally or dsRNA-
encoded toxins are active. In large-scale wine pro-
duction, fermentation is usually carried out using
defined S. cerevisiae starter cultures optimized for
fermentation performance and able to dominate
native yeasts in the grape must (Pretorius 2000).
To achieve the latter, wine and also sake fermen-
tation starter yeasts were engineered by cytoduc-
tion to possess the L-A and M viruses and the
corresponding killer phenotype (Ouchi and
Akiyama 1976; Hara et al. 1980; Seki et al. 1985;
Boone et al. 1990; Sulo and Michalcakova 1992;
Sulo et al. 1992; Michalcakova et al. 1994). Among
the S. cerevisiae dsRNA toxins, K2 was considered
to be most suitable for biocontrol in the wine
environment, since it is similar to K1 (see above)
122 Friedhelm Meinhardt and Roland Klassen
but displays higher activity at wine pH (3.5)
compared to K1 (Pfeiffer and Radler 1984). The
obtained industrial S. cerevisiae killer strains re-
tain desired flavour and fermentation character-
istics but are additionally able to suppress
indigenous S. cerevisiae strains due to toxin pro-
duction. Since K2 killer strains are frequent
among the natural population on grape surfaces,
the use of defined K2 killer-positive fermentation
starters which also display K2 immunity addition-
ally prevents overgrowth by the indigenous killer
(Jacobs and Van Vuuren 1991).
As an alternative to engineered killer strains,
fermentation starters which naturally express the
K2-type killer phenotype as well as desired fer-
mentation characteristics can be selected from the
population of indigenous yeasts (Lopes et al.
2007). However, non-Saccharomyces yeasts pres-
ent at grape surfaces are routinely insensitive to
the S. cerevisiae killer toxins (Young and Yagui
1978) and thus are largely restricting the biocon-
trol potential to Saccharomyces contaminants. Ad-
ditionally, indigenous non-Saccharomyces yeasts
often produce toxins, potentially enabling the pro-
ducer to overgrow the inoculated starter culture
and spoil the fermentation.
The killer toxin KpKt from K. phaffii displays
extensive activity against apiculate yeast species,
such as Hanseniaspora uvarum, which dominate
on grapes and grape juice and therefore KpKt was
analysed for its potential as a biopreservative
agent in wine (Ciani and Fatichenti 2001). During
experimental vinifications, KpKt was found to
display anti-H. uvarum activity comparable to
the routinely applied SO2, which is used to inhibit
apiculate yeasts at the prefermentation stage. It
was suggested that KpKt could substitute for
SO2, thereby eliminating undesired or harmful
residual traces of SO2 in the final product (Ciani
and Fatichenti 2001; Comitini et al. 2004b). The
killer toxins PiKt and KwKt from P. anomala and
K. wickerhamii were shown to be active and stable
in wine environment and are both capable of
inhibiting spoilage yeasts belonging to the genus
Dekkera/Brettanomyces, which can grow during
wine aging and represent a major problem due
to unpleasant odour and taste development
(Comitini et al. 2004a).
As several killer toxins, including those of P.
anomala, P. membranifaciens and D. hansenii are
not only active against yeast cells but also may
inhibit filamentous fungi, they are proposed as
potential agents for biocontrol of pre- and post-
harvest diseases caused by plant pathogenic fungi
(Walker et al. 1995; Santos and Marquina 2004b;
Santos et al. 2004). In particular, the toxin from P.
membranifaciens is active against Botrytis cinerea,
the causal agent of grey mould disease in grapes
and treatment of Vitis vinifera plants with either
purified toxin or the P. membranifaciens killer
strain protected against B. cinerea (Santos and
Marquina 2004b). The same toxin was also active
in suppressing B. cinerea growth on apples follow-
ing harvest, suggesting that P. membranifaciens
might be developed into a novel biocontrol agent
for grey mould disease (Santos et al. 2004). A killer
strain of P. anomala (YF07b) was selected from a
marine environment based on its ability to kill
Metschnikowia bicuspidate, a yeast species patho-
genic to Portunus trituberculatus, the most widely
fished crab species (Wang et al. 2007a, b). It was
suggested that the toxin of strain YF07b could be
used for controlling growth and infection of
the crabs by the pathogenic yeast (Wang et al.
2006a, b).
Silage, a fermentation product of field crops
and lactic acid bacteria used for feeding of rumi-
nant livestock is prone to aerobic spoilage by
lactic acid-utilizing yeasts. Two killer toxins were
analysed for their applicability in controlling si-
lage spoilage (Kitamoto et al. 1993; 1999; Lowes
et al. 2000). The K. lactis zymocin was found to be
active against a variety of silage spoilage yeasts
and the producer strain was further optimized for
biocontrol in silage by generating a zymocin-pro-
ducing phosphoenolpyruvate carboxykinase-neg-
ative mutant, which can no longer grow on lactic
acid and thus cannot contribute to spoilage (Kita-
moto et al. 1993, 1998). Co-inoculation of model
silage fermentations with the optimized K. lactis
strain and Lactobacillus plantarum indeed re-
duced aerobic spoilage and, thus, K. lactis was
suggested to be useful in prolonging the aerobic
stability of silage (Kitamoto et al. 1999). The other
toxin selected for control of silage and also dairy
product spoilage is HM-1 from Williopsis saturnus
var. mrakii, which exhibits a broad spectrum of
activity and exceptional stability as well as pH and
temperature stability (see above). The HM-1
(HMK)-encoding HMK gene was heterologously
expressed in Aspergillus niger and partially pur-
ified HM-1 was found to suppress aerobic spoilage
of model silage fermentations as well as growth of
Candia krusei and S. cerevisiae in yoghurt. Thus,
 Yeast Killer Toxins: Fundamentals and Applications
HM-1 is generally suited for the control of unde-
sired yeasts in both fermented feed and food pro-
ducts. Heterologous expression of HM-1 in lactic
acid bacteria might further extend the practicabil-
ity of such approaches (Lowes et al. 2000).
In order to protect plants against pathogenic
fungi and bacteria, the killer toxins KP4 and KP6
from Ustilago maydis and the synthetic killer pep-
tide KP (derived from P. anomala toxins; see
above) were heterologously expressed in tobacco
(Kinal et al. 1995; Park et al. 1996b; Donini et al.
2005). Authentic and active KP6 and KP4 could be
recovered from tobacco; however, the expression
level of KP6 was diminished compared to the
original toxin-producing U. maydis strain (Kinal
et al. 1995; Park et al. 1996b). KP4, in contrast, was
efficiently expressed; the expression was so effi-
cient that a small piece of leaf secreted enough
toxin to kill susceptible U. maydis (Park et al.
1996b). Therefore, heterologous expression of
KP4 was suggested to be potentially valuable for
the control of KP4 susceptible fungi in economi-
cally important crop plants (Park et al. 1996b).
By using a potato virus X-derived vector, the syn-
thetic killer peptide KP was heterologously
expressed in Nicotiana benthaminana as a fusion
to the viral coat protein (Donini et al. 2005). Such
purified chimeric virus particles displayed broad-
spectrum killer activity against human and
plant pathogens, as for KP (see above), and their
expression in planta conferred resistance against
plant-pathogenic Pseudomonas syringae, thereby
identifying KP as a promising molecule not
only for the treatment of fungal infections in
man (see above) but also to confer broad-
spectrum resistance to phytopathogens in plants
(Donini et al. 2005).
C. Killer Toxins in Biotyping
In 1983, it was realized that killer toxin sensitivity
patterns (sensitivity or insensitivity to a panel of
selected killer strains) can be used to distinguish
different Candida albicans isolates (Polonelli et al.
1983). Subsequently, the principle was extended
by the inclusion of more killer strains, the use of
partially purified toxins and computer-aided data
analysis, resulting in an improvement in repro-
ducibility (Polonelli et al. 1985; Buzzini and Mar-
tini 2000; Buzzini et al. 2003, 2007). Killer toxin
123
sensitivity patterns (KSPs) were shown to dis-
criminate not only different C. albicans strains
but also different species of other pathogenic
yeasts (Morace et al. 1984; Polonelli et al. 1985).
KSPs also proved useful for ecological studies on
the population diversity of indigenous wine yeasts
(S. cerevisiae; Sangorrin et al. 2002; Lopes et al.
2005). Since some yeast killer toxins are also ac-
tive against filamentous fungi or even bacteria
(see above), killer toxin-based biotyping has suc-
cessfully been extended to micro-organsims other
than yeast, including Penicillium camembertii,
Staphylococcus aureus, Neisseria meningitidis
and Mycobacterium tuberculosis (Morace et al.
1987, 1989; Polonelli et al. 1987).
However, as outlined by Buzzini et al. (2007),
the determination of KSP is currently not in routine
use for the differentiation of industrial or clinical
yeasts or other micro-organisms, mainly due to
problems associated with the limited reproducibili-
ty and automatization potential of phenotypic tests
involving living killer yeasts. Promising approaches
to achieve a higher reproducibility and discrimina-
tory power of KSP-based biotyping include the use
of a broader spectrum of killer toxins with known
function and their use as a standardized protein
preparation rather than as toxin-producing strains,
which would also enable automatization of the pro-
cedure (Buzzini et al. 2007). Further, the combina-
tion of KSP-based and molecular biotyping
methods, such as mtDNA-RFLP increases the dis-
ciminatory power of both and, thus, may represent a
promising perspective for the future application of
yeast killer toxins in biotyping (Lopes et al. 2005).
V. Concluding Remarks
The group of proteins subsumed as killer toxins
display considerable heterogeneity concerning
structure and evolutionary origin. Many are chro-
mosomally encoded and provide a selective ad-
vantage over sensitive non-killer yeasts in the
environment. In other cases toxin production is
conferred by viruses or virus-like elements, in
most of which the ability of toxin production is
genetically linked with specific immunity. In such
cases it can hardly be decided whether toxin pro-
duction evolved for the purpose of elimination
of competitors or for autoselection of the virus
or virus-like element. The fact that, at least for
124 Friedhelm Meinhardt and Roland Klassen
S. cerevisiae, such toxins display a narrow range of
activity, being essentially specific for non-virus-
carrying S. cerevisiae cells, may support an auto-
selective purpose of the toxins. Mechanistically, a
few toxic principles are repeatedly realized in
chromosomally and virally encoded killer toxins,
such as degradation or biosynthesis inhibition of
cell wall polysaccharides and direct membrane
permeabilization. In addition to lethal principles,
similar structures and biogenesis involving pro-
cessing of toxin precursors to mature toxins are
repeatedly encountered in toxins originating from
the chromosome or parasitizing viruses, despite
the fact that similarity in primary sequences is
lacking. Thus, convergent evolution may have re-
peatedly invented functionally similar toxins.
However, other lethal mechanisms, such as DNA
synthesis inhibition by the RNA virus-encoded
K28 toxin or tRNA-cleaving toxins encoded by
virus-like cytoplasmic dsDNA elements appear
unique to these elements yet. Nevertheless, only
a small fraction of recognized killer toxins has
been characterized in detail so far, raising the
possibility of repeated realization of DNA synthe-
sis-inhibiting or tRNA-cleaving activities or even
further yet unknown toxic mechanisms in other
killer yeasts. As for the antibiotics which are the
constituents of a similar antagonistic interaction,
killer toxins may potentially be developed into a
new generation of antifungal agents or find other
useful applications in the food and feed industry.
Acknowledgement. Financial support by the Deutsche
Forschungs Gemeinschaft (DFG) grant no. ME 1142/5-1
is gratefully acknowledged.
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7 Evolutionary and Ecological Interactions of Mould and Insects
MARKO ROHLFS1, MONIKA TRIENENS1, ULRIKE FOHGRUB2, FRANK KEMPKEN2
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Genetics of Secondary Metabolites in
Mycelial Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Different Types of Fungal Secondary
Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Non-Ribosomal Peptides . . . . . . . . . . . . . .
3. Polyketides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Terpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Fungal Secondary Metabolite Clusters . . . .
1. Aflatoxin and Sterigmatocystin
Clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Epipolythiodioxopiperazines . . . . . . . . . .
C. Regulation of Secondary Metabolite
Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Pathway-Specific Regulation . . . . . . . . . . .
2. Global Regulation . . . . . . . . . . . . . . . . . . . . . .
3. Regulation by Signal
Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Activation of Silent Secondary
Metabolite Clusters . . . . . . . . . . . . . . . . . . . . .
III. Three Types of Fungus–Insect Interactions
and the Role of Secondary Metabolites . . . . . . .
A. Host–Pathogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Predator–Prey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Interspecific Competition . . . . . . . . . . . . . . . . . .
IV. Melding Ecology and Molecular Biology . . . . . .
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. Introduction
Besides plants, fungi are among the most prodi-
gious producers of secondary metabolites, of
which some belong to the most toxic compounds
in the living world. In contrast to plants, however,
1
Zoological Institute, Department of Animal Ecology,
Christian-Albrechts-University of Kiel, Olshausenstrasse 40,
24098 Kiel, Germany
2
Botanical Institute, Department of Plant Genetics and
Molecular Biology, Christian-Albrechts-University of Kiel,
Olshausenstrasse 40, 24098 Kiel, Germany; e-mail: fkemp-
ken@bot.uni-kiel.de
where the function of many secondary metabolites
131 is regarded as a direct defence against pathogens
and herbivores that reduce their survival and re-
132 production (Steppuhn et al. 2004), the benefits of
producing low molecular weight compounds to
132
132 fungi are often obscure. In contrast to this critical
132 gap in knowledge about fungal biology, an enor-
133 mous research effort world-wide has been devoted
133 to identifying biochemical pathways and the
133 underlying genetic mechanisms leading to the
133 biosynthesis of fungal secondary metabolites,
135 especially those with detrimental (e.g. toxins) or
beneficial (e.g. antibiotics) impact on humans and
135 animals (Keller et al. 2005; Hoffmeister and Keller
135 2007). Although molecular biologists have just
135
scratched the surface of secondary metabolism, fila-
138 mentous fungi, such as the model genus Aspergillus,
appear to have evolved sophisticated ways of pro-
139 ducing and regulating a plethora of secondary meta-
bolites (Bok and Keller 2004; Yu and Keller 2005;
139
140 Herrmann et al. 2006; Perrin et al. 2007; Shwab et al.
141 2007). The ability to regulate secondary metabolism
143 has often been suggested to reflect an evolutionary
146 adaptation to ensure efficient exploitation of envi-
147 ronmental resources and to synthesise the energeti-
147
cally costly compounds only when the ecological
conditions demand the employment of these ‘chem-
ical weapons’ against their natural enemies and
competitors (Vining 1990; Demain and Fang
2000). Therefore, chemical diversity and its regula-
tion in fungi may be considered as a key life-history
trait that is favoured by natural selection because it
ensures survival and reproduction in a variable en-
vironment. In this review, we focus on the role
of fungal secondary metabolites in interactions
with antagonistic insects. In particular, we address
the question when, how and why should fungi
use secondary metabolites as adaptive responses
to interactions within a fungus natural environ-
ment, and we hope to stimulate cross-disciplinary
communication among ecologists, mycologists and
molecular biologists.
Physiology and Genetics, 1st Edition
The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
132 Marko Rohlfs et al.
A number of chapters in this volume also
cover mycotoxins or deal with related subjects
in more detail: Cyclic peptides and depsipeptides
from fungi by Heidrun Anke, non-ribosomal
peptide synthetases of fungi by Kathrin Eisfeld,
physiological and molecular aspects of ochra-
toxin A biosynthesis by Rolf Geisen and Markus
Schmidt-Heydt, and polyketide biosynthesis in
fungi by Julia Schümann and Christian Hert-
weck.
II. Genetics of Secondary Metabolites
in Mycelial Fungi
A. Different Types of Fungal Secondary
Metabolites
Many if not all fungi produce specific secondary
metabolites which are not required for primary
metabolism. These include toxins like aflatoxins
or fumonisins and pharmaceutically useful drugs
such as penicillin, cephalosporin or cyclosporine
(Cole and Schweikert 2003). Without doubt, many
fungal secondary metabolites have not yet been
detected and further efforts to discover new fungal
secondary metabolites may provide important
drugs in the future. Secondary metabolites are
species-specific and often accumulate in certain
stages of development or during specific morpho-
genic differentiation (Bennett and Bentley 1989).
Usually fungi can survive without secondary
metabolites, at least in the laboratory. However,
secondary metabolites may be of vital importance
to the fungus in its particular ecological niche. A
recently performed survey included some 1 500
Table 7.1. Some important secondary metabolites
Main group Secondary metabolite
Alkaloids Ergotamine
Peptides Gliotoxin
Cyclosporin
Polyketides Aflatoxin B1
Lovastatin
Terpenes Gibberellin GA3
secondary metabolites and provided evidence, that
more than 50% of the metabolites showed antibac-
terial or antifungal activities. Some even showed
antitumor activity (Pelaez 2005). The latter may be
due to the fact that antifungal molecules may also
harm other eukaryotic cells, including tumour cells.
Fungal secondary metabolites may be separated
in four groups, i.e. alkaloids, non-ribosomal pep-
tides, polyketides and terpenes. Examples of some
well characterized metabolites are given in Table 7.1.
1. Alkaloids
Indole alkaloids are most often synthesized from
dimethylallyl pyrophosphate and tryptophan.
Sometimes other amino acids may be involved.
The ergotamine pathway of Claviceps purpurea is
particularly well characterised. The first enzymatic
step is prenylation of tryptophan by the dimethyl-
allyl trypthophan synthetase, followed by methyl-
ation of dimethylallyl tryptophan and several
oxidation steps leading to lysergic acid. By the
action of two different non-ribosomal peptide
synthetases finally ergotamine is synthesised
(Tudzynski et al. 1999, 2001b).
2. Non-Ribosomal Peptides
A database of non-ribosomal peptides of fungal
and bacterial origin was established and published
recently (Caboche et al. 2008). These molecules are
synthesized by a class of multidomain and multi-
module fungal enzymes called non-ribosomal
peptide synthetases, which catalyse a variety of
non-ribosomal peptides using proteinogenic and
non-proteinogenic amino acids. Each module in a
Organism References
Claviceps purpurea (Haarmann et al. 2005)
Aspergillus fumigatus (Keller et al. 2005;
Bok et al. 2006a;
Fox and Howlett 2008)
Tolypocladium inflatum (Hoppert et al. 2001;
Keller et al. 2005)
Aspergillus flavus (Katz and Donadio 1993;
Keller et al. 2005)
Aspergillus terreus (Keller et al. 2005;
Collemare et al. 2008)
Gibberella fujikuroi (Keller et al. 2005;
Appleyard et al. 1995;
Kawaide 2006)
 Evolutionary and Ecological Interactions of Mould and Insects
non-ribosomal peptide synthetase has several
domains for recognition, activation and covalent
binding of a module-specific amino acid. The di-
versity of non-ribosomal peptides is based on
varying length of peptides, linear or cyclic peptides
and variations in the function of enzyme domains
(Keller et al. 2005a). The first non-ribosomal pep-
tide synthetase which has been characterised in
detail catalyses the formation of b-lactam antibio-
tics (Smith et al. 1990). The immuno-suppressive
drug cyclosporine, acyclic undecapeptide, is also
synthesized by non-ribosomal peptide synthetase
in the filamentous fungus Tolypocladium inflatum
(Zocher et al. 1986; Weber et al. 1994). Non-ribo-
somal peptide synthetases are also discussed with
respect to fungal pathogenicity (Collemare et al.
2008).
3. Polyketides
Polyketides resemble the most abundant fungal
secondary metabolites. Well known examples are
aflatoxins (Sweeney and Dobson 1999) and the
anti-cholesterol substance lovastatin (Kennedy
et al. 1999). Fungal polyketides are synthesized
by type I polyketide synthases (PKS) which are
multidomain enzymes similar to eukaryotic fatty
acid synthases. Three domains are essential in all
fungal PKS, i.e. acyl carrier, acyltransferase, and
ketoacyl CoA synthase, while other domains such
as enoyl reductase, dehydratase, or ketoreductase
may or may not be present. Like fatty acid
synthases, fungal PKS condense short-chain car-
boxylic acids (acetylCoA, malonylCoA) to form
carbon chains of different length. However, while
fatty acid synthases fully reduce b-carbon, this is
optional with fungal PKS. The diversity of fungal
polyketides is due to varying iteration reactions,
varying number of reduction reactions, type of
extender units used, and a variety of post-polyke-
tide synthesis steps (Keller et al. 2005a).
4. Terpenes
Terpenes are typically associated with plants.
Consequently a vast amount of terpenes are
known from plants. However, there are also
fungi which produce terpens. Several fungal ter-
pene cyclases have been identified and charac-
terised, i.e. from Gibberella fujikuroi (Tudzynski
et al. 2001a) or a Fusarium species (Rynkiewicz
et al. 2001). All terpenes are made of several
133
isoprene units, can be linear or cyclic, saturated
or unsaturated, and may be modified in several
ways.
B. Fungal Secondary Metabolite Clusters
In bacteria, genes encoding enzymes of a specific
pathway are usually combined in operons. This
ensures a rather simple regulation which allows
for expression or repression of all pathway specific
genes at the same time. Although thought to be res-
tricted to bacteria, similar structures have been
observed in fungi. Secondary metabolite genes typi-
cally are organised in gene clusters (Hull et al. 1989;
Keller and Hohn 1997). In this review we give an
overview of two important secondary metabolite
clusters and their regulation. In addition a compi-
lation of secondary metabolite gene classes in
selected Aspergillus species are shown in Table 7.2.
1. Aflatoxin and Sterigmatocystin Clusters
The aflatoxin/sterigmatocystin gene clusters are
particularly well characterised. While in Aspergil-
lus nidulans only sterigmatocystin is produced,
in A. flavus and A. parasiticus sterigmatocystin is
the penulatimate precursor to aflatoxin synthesis.
The biosynthesis of sterigmatocytin and aflatoxin
is shown in Fig. 7.1 (Hamasaki et al. 1973; Barnes
et al. 1994; Hicks et al. 2002; Yu et al. 2004). The
aflatoxin gene cluster has a size of about 70 kb,
and 21 genes of this cluster have been function-
ally characterized in more detail, while the func-
tions of six other genes have yet to be elucidated
(Yu et al. 2004). Most of the functionally
assigned genes encode different biosynthetic
enzymes (Yu and Keller 2005). One gene, aflR,
Table 7.2. Secondary metabolite gene classes in selected
Aspergillus species (modified from Keller et al. 2005b)
Gene A. A. A.
fumigatus nidulans oryzae
DMATS (dimethylallyl 7 2 2
tryptophan synthetase)
FAS (fatty acid synthase) 1 6 5
NRPS (non-ribosomal 14 14 18
peptide synthetase)
PKS (polyketide synthase) 14 27 30
SesCyc (sesquiterpene 0 1 1
cyclase)
134 Marko Rohlfs et al.

HO O HO O HO O HO OH
Acetyl CoA
Malonyl CoA
stcE
stcA HO OH HO OH
stcJ O Norsolorinic Acid (NOR) O Averantin (AVN)
stcK
Polyketide Procursor stcF
OH O OH HO O OH
O
stcG? O
HO O CH3 HO OH CH3
O Averufin (AVF) O Averufannin (AVNN)
stcB or stcW
OH O OH HO O OH
stcB or stcW O

HO O OH O OH O CH3
HO
O O O Versiconal hemiacetal Acetate (VHA)
Hydroxyversicolorone (HVN)
stcI?
HO O OH

HO O OH OH stcN?
HO O OH O Versiconal (VAL) HO O OH

stcL
HO O O Branch A O O
O Versicolorin A (VERA) HO
O Versicolorin B (VERB)

Branch B
stcS, stcU
O OH
O OH
O
O
O O OH
O O OH Dihydro-demethylsterigmatocystin (DHDMST)
Demethylsterigmatocystin (DMST)
stcP
O OH
O OH O
O OCH3
O O
O O OCH3 Dihydrosterigmatocystin (DHST)
sterigmatocystin (ST)

O OCH3
O OCH3
O
O
O O OCH3
O O OCH3 O O Dihydr-o-methylsterigmatocystin (DHOMST)
O -methylsterigmatocystin (OMST) O O
O
O

O O OCH3
Aflatoxin B1(AFB1) O O OCH3
Aflatoxin B2(AFB2)

Fig. 7.1. Aflatoxin/sterigmatocystin biosynthetic pathway. Figure modified from Hicks et al. (2002) with permission
 Evolutionary and Ecological Interactions of Mould and Insects
encodes a binuclear zinc cluster transcription
factor (Chang et al. 1993; Woloshuk et al. 1994;
Yu et al. 1996a; Keller and Hohn 1997; Shimizu
et al. 2003), which is specific for fungi (Keller
and Hohn 1997; Fernandes et al. 1998; Yu and
Keller 2005). The regulatory functions of these
genes are discussed below. The sterigmatocystin
cluster of A. nidulans is somewhat smaller (60
kb), but still contains 25 genes (Hicks et al.
2002). A comparison of the two gene clusters is
given in Fig. 7.2. Homologous genes in both
clusters are shown in the same colours. In addi-
tion Table 7.3 gives a compilation of all genes in
the two clusters, their accession numbers and
encoded enzymes.
2. Epipolythiodioxopiperazine clusters
Epipolythiodioxopiperazines are toxic second-
ary metabolites, almost exclusively produced
by ascomycetes. This class of secondary meta-
bolites is characterised by the presence of a
sulfur-bridged dioxopiperazine ring synthesised
from two amino acids (see Fig. 7.3). Toxicity
results from cross-linking proteins via cysteine
residues and generating reactive oxygen
species through redox cycling (Fox and Howlett
2008). So far 14 different epipolythiodioxopip-
erazines are known, with gliotoxin produced by
Aspergillus fumigatus, A. flavus, or A. niger
being the best characterized example (Fox and
Howlett 2008). Another important epipolythio-
dioxopiperazines is sirodesmin PL from Lepto-
sphaeria maculans, which is involved in plant
pathology and causes necrosis on plants
(Rouxel et al. 1988). The biosynthesis routes of
sirodesmin PL and gliotoxin were deduced dec-
ades ago by the use of labelling experiments and
analysis of intermediates (see literature cited by
Fox and Howlett 2008). The pathway for glio-
toxin biosynthesis is shown in Fig. 7.4. More
recently the gene clusters for both metabolites
were characterized (Fig. 7.2). The sirodesmin
cluster was characterised first (Gardiner et al.
2004). Then, based on sequence comparison of
the L. maculans sirodesmin genes the gliotoxin
gene cluster was predicted for A. fumigatus
(Gardiner and Howlett 2005). This prediction
was confirmed by knock-out experiments
(Cramer et al. 2006; Kupfahl et al. 2006).
135
C. Regulation of Secondary Metabolite
Synthesis
1. Pathway-Specific Regulation
As mentioned above, aflR encodes a transcription
factor. Both, sterigmatocystin and aflatoxin clusters
contain an aflR gene. This gene is essential for
expression of all other cluster genes and is post-
transcriptionally regulated (Yu et al. 1996a). Fur-
thermore, the AflR protein is necessary for the
positive co-regulation of AF/ST biosynthetic gene
expression (Feng and Leonard 1998; Meyers et al.
1998; Hicks et al. 2002; Shimizu et al. 2003; Price
et al. 2006). Deletion or mutation of aflR led to
strong down regulation of sterigmatocystin and
aflatoxin gene expression. A second gene aflJ also
is involved in gene regulation, although it does not
encode a typical transcription factor. Deletion of
aflJ results in a failure to produce aflatoxin, and
certain pathway intermediates cannot be used by
the fungus when applied exogenously. However
some transcripts of the pathway genes still accumu-
late and therefore aflJ does not appear to influence
aflatoxin regulation on a transcriptional level
(Meyers et al. 1998; Yu and Keller 2005; Du et al.
2007). Likewise the gliZ gene, a ZN(II)2Cys6 (see
Fig. 7.2) regulates gene expression of the gliotoxin
gene cluster, as an A. fumigatus mutation in this
gene led to the unability to produce gliotoxin (Bok
et al. 2006a). This mutant is also characterised by a
reduced virulence.
2. Global Regulation
Secondary metabolite biosynthesis is influenced
by a number of environmental factors such as
nitrogen or carbon sources, temperature or light.
There is evidence that such environmental signals
are mediated through general global transcription
factors, e.g. for carbon CreA and for nitrogen
AreA (Ehrlich et al. 2003; Mihlan et al. 2003).
Several secondary metabolite gene clusters are
positively or negatively regulated by these tran-
scription factors.
In Aspergillus species a novel nuclear pro-
tein, LaeA, was discovered as a global regulator
of secondary metabolism. Deletion mutants have
lost expression of metabolic gene clusters, in-
cluding the sterigmatocystin, lovastatin and
penicillin clusters. Over-expression of laeA
increases transcription and subsequent product
 136

A1
norB cypA aflT pskA hypBl nor -l hexA hexB aflR aflJ adhA estA norA ver-1 verA avnA verB hypB2 omtB avfA omtA ordA vbs cypX maxY ordB hypA nadA

A2
pskA cypX nor- l aflR aflJ avnA adhA estA hexA hexB verB hypB2 vbs avfA omtB ordB hypA verA ver- l norA moxY

B3
sirT sirD sirO sirN sirA sirZ sirS sirE sirM sirC sirG sirP sirB sirR sirJ sirl sirQ sirH
Marko Rohlfs et al.

B4
gliZ gliI gliJ gliP gliC gliM gliG gliK gliA gliN gliF gliT

Fig. 7.2. Secondary metabolite gene clusters: A1 aflatoxin gene cluster from
A. flavus; A2 sterigmatocystin gene cluster from A. nidulans; homologous
genes in A1 and A2 are given in the same colour. Gene names and encoded
peptides are given in Table 7.3. Figure modified from Ehrlich et al. (2008) with
permission. B3 Leptosphaeria maculans sirodesmin PL; B4 Aspergillus fumigatus
gliotoxin biosynthetic gene clusters. Common ETP genes include non-ribosomal
peptide synthetase (P), thioredoxin reductase (T), methyl transferases (M, N),
glutathione S-transferase (G) and cytochrome P450 mono-oxygenase (C), amino
cyclopropane carboxylate synthase (ACCS; I), dipeptidase (J), as well as a
transcriptional regulator (Z) and a transporter (A). Other genes do not have
homologues in the other cluster and encodes changes in the side chains of the
ETP molecule structure, including cytochrome P450 mono-oxygenases (F, B, E),
a prenyl transferase (D), an acetyl transferase (H), epimerases (Q, S, R), an
oxidoreductase (O) and a hypothetical protein (K; Gardiner and Howlett 2005).
Genes shaded in grey encode proteins with best matches to proteins with no
potential roles in ETP biosynthesis. The forward slash marks represent a 17-kb
region of repetitive DNA. Homologous genes in B3 and B4 are given in the same
colour. Figure modified from Fox and Howlett (2008) with permission
 Evolutionary and Ecological Interactions of Mould and Insects 137

Table 7.3. Comparison of aflatoxin and sterigmatocystin pathway gene

Gene Original name and other names used ST gene Enzyme or product
(accession no.)a homologueb
aflA fas-2 (hexA; AF391094) stcJ FAS alpha subunit
aflB fas-1 (hexB; AF391094), uvm8, fas1, fas-1A stcK FAS beta subunit
(L48183)
aflC pksA (Z47198), pksL1 (L42765, L42766) stcA PKS
aflD nor-1 (L27801) stcE Reductase
aflEc norA, aad (U24698), adh-2 in A. flavus (U32377) stcV NOR reductase/dehydrogenase
aflFc norB Dehydrogenase
aflG avnA (U62774), ord-1 (L40839) stcF P450 monooxygenase
aflH adhA (U76621) stcG Alcohol dehydrogenase
aflI avfA (AF154050), ord-2 (L40840), (AF159789 in stcO Oxidase
A. flavus)
aflJ estA (AF417002) stcI Esterase
aflK vbs (AF169016, U51327) stcN VERB synthase
aflL verB (AF106958), (AF106959 and AF106960 in stcL Desaturase
A. flavus)
aflM ver-1 (M91369) stcU Dehydrogenase/ketoreductase
aflNc verA stcS (verA) Monooxygenase
aflO dmtA (mt-I) (AB022905, AB022906), omtB stcP O-methyltransferase I or
(AF154050), (AF159789 in A. flavus) O-methyltransferase B
aflP omtA (L25834), omt-1 cDNA (L22091), (L25836 O-methyltransferase A or
in A. flavus) O-methyltransferase II
aflQ ordA (AF017151, AF169016), A. flavus ord-1 Oxidoreductase/P450 monooxygenase
(U81806, U81807)
aflR aflR (L26222), apa-2 (L22177), afl-2, (AF427616, aflR Transcription activator
AF441429)
aflS aflJ (AF002660), (AF077975 in A. flavus) Unnamed Transcription enhancer
aflT aflT (AF268071) Transmembrane protein
aflU cypA P450 monooxygenase
aflV cypX (AF169016) stcB P450 monooxygenase
aflW moxY (AF169016) stcW Monooxygenase
aflX ordB stcQ Monooxygenase/oxidase
aflY hypA Hypothetical protein
aflR2d aflR2 (AF452809) Second copy Transcription activator
aflS2 aflJ2 (AF452809, AF295204) Second copy Transcription enhancer
aflH2 adhA2 (AF452809) Second copy Alcohol dehydrogenase
aflJ2 estA2 (AF452809) Second copy Esterase
aflE2 norA2 (AF452809) Second copy Dehydrogenase (early terminated)
aflM2 ver-1B (AF452809) Second copy Dehydrogenase (missing N-terminal)
aflO2 omtB2 (AF452809) Second copy Methyltransferase B (missing N-terminal)
a
The accession number of the complete 82 081-bp aflatoxin gene cluster, including a sugar utilization gene cluster, in A. parasiticus
is AY391490 and updates the sequences of the underlined accession numbers. The genes and their accession numbers are from
A. parasiticus unless otherwise noted.
b
The accession number of the ST gene cluster in A. nidulans is U34740, and the corresponding contig number is 1.132 (from 183 018 to
242 843) in the Whitehead database.
c
The placements of aflE (norA), aflF (norB), and aflN (verA) in the pathway were based on their homologies to aflatoxin or ST genes and
their functions have not been experimentally confirmed.
d
The aflR2, aflS2, aflH2, aflJ2, aflE2, aflM2, and aflO2 genes are partially duplicated cluster genes (second copy) in A. parasiticus, and
their functions and chromosomal locations in the genome have not yet been clarified.
e
Arrows signify conversion.

formation (Bok and Keller 2004). The laeA gene
is negatively regulated by AflR and also regulated
by two signal transduction factors, protein kinase
A and RasA. Interestingly, spore formation in
delta laeA is rather similar to the wild type,
indicating a primary role of LaeA in regulation
of metabolic gene clusters (Bok and Keller 2004).
LaeA is also known to regulate virulence in A.
fumigatus, as deletion mutants show reduced
virulence (Bok et al. 2005) and reduced gliotoxin
levels (Sugui et al. 2007). Genes in 13 of 22
secondary metabolite clusters appear to be
138 Marko Rohlfs et al.

Fig. 7.3. Structure of epipoly- a b

O
thiodioxopiperazines. A Generic O
structure of an epidithiodioxop- R3
R1 N
iperazine (ETP). R = any atom or N S-S
group. B Gliotoxin S-S N
N OH
R2 OH
O
O OH

O NH2 proteins, VeA and VelB, in which VeA bridges

OH HO
OH VelB to LaeA. Deletion in either VeA or VelB
NH2 O
results in defects in both, sexual development
Phenylalanine Serine
and secondary metabolites. VeA is light-regu-
peptide synthetase (GliP)
lated. The VeA-VelB-LaeA complex is formed in
the dark only and significantly increases second-
ary metabolism (Bayram et al. 2008). In addition
O
to LaeA an histone deacetylase (HDAC) appears
NH to be involved in epigenetic regulation of second-
cyclo-(L-phenylalanyl-L-
HN
OH
seryl) ary metabolite expression (Shwab et al. 2007).
While positive regulation of Aspergillus second-
O
ary metabolite clusters by LaeA has been shown
sulfurisation to be less spatially limited, suppression of sec-
dithiol oxidation thioredoxin reductase ondary metabolite biosynthesis by expression of
(GLliT) hdaA is confined to telomere-proximal clusters
O
that encode sterigmatocystin and penicillin
S S
NH (Shwab et al. 2007). Such different regulatory
HN OH mechanisms able to independently activate or
O disable secondary chemical production may
provide fungi with an efficient means of adap-
oxidation tively responding to multiple natural enemies
methylation methyltransverases (GliM or GliN) and competitors.
O

CH3
N
S S
N OH
OH O Secondary metabolite synthesis and regulation of
Gliotoxin
Fig. 7.4. Biosynthesis of gliotoxin. Figure modified from
Fox and Howlett (2008) with permission
regulated by LaeA (Perrin et al. 2007). Most
interestingly, LaeA regulation of the sterigmato-
cystin cluster appears to be location-specific.
Genes next to the cluster are not affected by
LaeA, but genes artificially introduced into the
cluster are subject to LaeA regulation. LaeA
appears to be a methyltransferase which may
directly interact with chromatin structure (Bok
et al. 2006a). Recently it was shown that
LaeA forms a heterotrimeric complex with two
3. Regulation by Signal Transduction
secondary metabolite gene clusters are also con-
trolled by upstream signalling mechanisms
(Yu and Keller 2005). As shown in Fig. 7.5, a
complex pathway controls vegetative growth, con-
idiation, and production of secondary metabolites
in A. nidulans, in which heterotrimeric G protein
and its regulation are of crucial importance. Ex-
ternal signals at a seven transmembrane receptor
lead to the dissociation of Ga from the heterotri-
meric G protein. As in other fungi, three Ga genes
are present in Aspergillus species. One Ga,
encoded by FadA, activates the penicillin biosyn-
thesis and via cAMP and protein kinase A induces
hyphal growth. Gbg subunits inhibit asexual
development. The G protein regulator FlbB plays
a key role by converting Ga-GTP to Ga-GDP.
 Evolutionary and Ecological Interactions of Mould and Insects
developmental genes
FluG SFGs ? brlA
flbE, flbD, flbB, flbC
?
? ?
FlbB
RGS
Pi
GDP
? Gβ
FadA
GpgA
Gα
Gγ
SfaD
Gβ GTP GDP
penicillin
biosynthesis
Fig. 7.5. Antagonistic regulation by signal transduction of
vegetative growth, conidiation and production of second-
ary metabolites. AflR Sterigmatocystin transcription fac-
tor. brl A, fluG, flbE, flbD, flB, flbC Genes required for
Activation of asexual development requires par-
tial inhibition of the G protein-mediated signal-
ling. This requires activity of fluG and flbA (Yu
et al. 1996b; Hicks et al. 1997; Rosen et al. 1999;
Tag et al. 2000). Protein kinase A induces hyphal
growth and inhibits both asexual development
and sterigmatocystin biosynthesis (Shimizu et al.
2003). The latter is due to inhibition of LaeA and
AflR. For a detailed review see Yu and Keller
(2005). Signal transduction will also be necessary
to coordinate and regulate other secondary me-
tabolite genes in response to stresses and confron-
tation with other micro-organisms or antagonistic
insects. Yet literally nothing is known about this
type of regulation.
4. Activation of Silent Secondary Metabolite
Clusters
Genomic analysis has led to the identification of a
large number of secondary metabolite genes and
gene clusters (some included in Table 7.2). How-
ever, many of these clusters remain silent under
laboratory conditions. This may be due to a lack
of appropriate signals which will lead to cluster
expression under certain natural conditions.
Recently, a silent gene cluster was activated
by artificially expression of its regulatory gene
(Bergmann et al. 2007). While this approach will
139
asexual
development
GpgA
Gγ
SfaD
PkaA
hyphal
GTP PkaA growth
FadA
Gα
LaeA AflR ST production
conidiation (Lee and Adams 1996). LaeA Global secondary
metabolite regulator, PkaA protein kinase A, RGS regula-
tor of G protein signaling. Figure modified from Yu and
Keller (2005) with permission
be useful for discovery of new drugs (Brakhage
et al. 2008), it will also be useful to identify the
natural signals which led to activation of silent
clusters. This may be caused by interaction
with specific chemicals, or by contact with other
micro-organisms or natural enemies, such as
insects (see below).
III. Three Types of Fungus–Insect
Interactions and the Role
of Secondary Metabolites
There are three basic effects of species interaction:
(1) one species may cause an increase in the sur-
vival, growth and fecundity of another species, (2)
it may cause a decrease, or (3) it may have no
effect at all. The most obvious example of the
second type of interaction (in symbolic form: ‘+
’) is a predator–prey relationship, that is one
species is at least partly eaten by the other. Besides
host-parasite interactions and herbivory, fungiv-
ory also falls in this category. In addition to com-
mensalism (+ 0), amensalism ( 0), and
mutualism (+ +), which are probably only minor
categories, interspecific competition (– –) is
the most prevalent form of species interaction.
The ‘– –’ symbolism stresses its essential aspect,
140 Marko Rohlfs et al.
namely that two species cause a demonstrable
reduction in each other’s fitness parameters (i.e.
survival, growth, and/or reproduction). Neverthe-
less, it should be noted that whenever two species
compete there are some conditions under which
one species is significantly more affected than the
other. For instance, the dominance relationship
between two competitors is altered when there is
a temporal variation between the settlement of the
two species in a habitat. In general, species that
become established earlier have a greater chance
of being competitively superior over later arrivals.
These historical effects are called priority effects
and could result from size- or stage-dependent
competition. These detrimental effects of inter-
specific competition can be expected to act
through some combination of reproductive suc-
cess and survival. It can be expected that there is a
competition for a resource that is limited (e.g.
food supply), that the effects will be density-de-
pendent, and that this type of interaction is recip-
rocal (Begon et al. 1996).
In this review, we concentrate on antagonistic
interactions between fungi and insects and high-
light two types of the ‘+ -’ interrelationship, name-
ly: (1) where fungi are pathogens of insects and (2)
where fungi are a nutritional source for arthro-
pods. Special attention is paid to the competitive
interplay (– –) between saprophytic fungi and
saprophagous insects since competition between
distantly related organisms has repeatedly been
suggested to be widespread but has only rarely
been addressed in ecological research and is vir-
tually absent from functional fungal and insect
molecular biology.
A. Host–Pathogen
Pathogens are considered as biological agents that
cause suffering of animal hosts upon infection.
This definition is medical rather than biological,
i.e. an evolutionary definition, because it states
nothing about the reproductive success of the
pathogenic organism invading an animal’s body.
For example, filamentous fungi, such as Aspergillus
fumigatus, may be serious vertebrate patho-
gens, though this type of host is assumed to be a
‘dead end’ since fungi are unable to reproduce and
disperse to new hosts (Tekaia and Latgé 2005).
Therefore, the evolution of virulence factors
responsible for causing, e.g. aspergillosis, are
unlikely to have taken place in vertebrates and it
is still unknown what kind of selective forces have
turned opportunistic Aspergillus species into
dreaded human pathogens. In contrast, many
mould-like fungi have been selected for success-
fully exploiting insects as hosts (Vega and
Blackwell 2005). After germination of spores on the
insect’s cuticle, fungal hyphae start penetrating
the cuticle, followed by invasion of epidermis
and hypodermis. Insect death is the consequence
of tissue invasion by hyphae or hyphal bodies
proliferating in the insect’s hemolymph (Clarkson
and Charnley 1996). However, fungi, after having
successfully conquered the exoskeleton as a first
line of insect defence, are additionally faced with
the challenge to overcome insect cellular and
humoral immune responses before being able to
grow in their hosts (Gillespie et al. 2000; Gottar
et al. 2006). Finally, the dead insect body allows
saprotrophic growth and fungi burst the insect’s
exoskeleton, which eventually leads to the forma-
tion of reproductive organs and spores which
are dispersed by wind or phoresy. As recently
summarised by Furlong and Pell (2005), entomo-
pathogenic fungi seriously impact trophic inter-
actions within insect communities and hence
biological control programmes.
Interestingly, there is a huge variation among
and within different fungus species in the ability
to infect hosts, which may be attributed to spe-
cies- or strain-specific differences in the ability to
express virulence factors (Clarkson and Charnley
1996), but additionally depends on the host’s im-
mune system and hence the ability to defend one-
self against the fungal invader (Tinsley et al. 2006).
Given inherited variation in virulence, insect hosts
may represent a selective environment that
favours different degrees of expression of viru-
lence factors, such as immunosuppressive agents
like some mycotoxins (see below), which might
also be of relevance when fungi enter vertebrate
‘hosts’ (Reeves et al. 2004). It should be noted,
however, that well adapted entomopathogenic
fungi (e.g. Beauveria bassiana) are not among
the most prominent vertebrate pathogens, though
invasive infection of humans is possible (Henke
et al. 2002). This may indicate that pathogen
evolution in arthropods does not correlate with
pathogenicity in vertebrates, i.e. virulence appears
to be based on specific components required
for infection of either insect or vertebrate
hosts, rather than on non-specific components.
 Evolutionary and Ecological Interactions of Mould and Insects
Identification of the evolutionary origin of viru-
lence factors may become even more difficult
when we consider the fact that opportunistic
pathogens, such as Aspergillus flavus or A. fumi-
gatus, but also facultative entomopathogenic
fungi, can perform vital growth and reproduction
as saprophytes. While fungi have to overcome
passive and active defence mechanisms in insect
hosts, decaying organic matter may be occupied
by a large number and high density of competing
micro-organisms (yeast, bacteria, other filamen-
tous fungi) and fungi are likely to encounter an-
tagonistic insects that can seriously hamper
fungal growth (see Sect. III.C); thus, in compari-
son to living hosts, decaying organic matter poses
a different ecological challenge to the fungi. Inter-
estingly, the first data point in possible trade-offs
between a saprophytic and a pathogenic life style
is A. flavus (Scully and Bidochka 2005), i.e. there
may be constraints on being equally successful in
both environments. The latter comprises the ideas
of the life history paradigm, namely that organism
cannot simultaneously maximise all components
of fitness (Stearns 1992). As suggested by the
results of the study by Scully and Bidochka
(2005), improvement of the pathogenic lifestyle
was paid for with a decrease in the ability to
exploit dead organic material. Nonetheless, com-
prehensive studies addressing the extent to which
selection pressures in these different environ-
ments are affecting virulent and saprobic capaci-
ties in fungi remain to be carried out.
Toxic secondary metabolites have often been
suggested to constitute important virulence fac-
tors enabling successful colonisation of insect
hosts by fungi (Clarkson and Charnley 1996),
such as destruxins or beauvericin from the potent
entomopathogens Metarhizium anisophilae and
Beauveria bassiana, respectively. Recently, pro-
duction of gliotoxin, immunosuppressive agent
in vertebrates (Bok et al. 2005), has been corre-
lated with virulence of Aspergillus fumigatus in
the greater wax moth Galleria mellonella (Reeves
et al. 2004); and the injection of aflatoxin B1 into
larvae of the Egyptian cotton leaf worm (Spodop-
tera littoralis) had a huge impact on insect devel-
opment (Abdou et al. 1984). However, given that
the arthropod exoskeleton provides an efficient
physical barrier against pathogens, several studies
imply that selection for virulence of entomopatho-
genic fungi is related to the proteinase and chit-
inase secretion required to degrade the insect
141
cuticle (St Leger et al. 1997; Bagga et al. 2004;
Wang et al. 2005; Gottar et al. 2006; Fan et al.
2007). Yet the production of toxic metabolites
may become important when the fungus has
successfully entered the insect’s body cavity
(Wang et al. 2005), e.g. to impair the host’s im-
mune response. Thus, for opportunistic as well as
for facultative entomopathogenic fungi, the causal
relationship between toxic secondary metabolite
expression and virulent phenotypes still remains
elusive. However, studies using an experimental
evolutionary approach, similar to the work by
Scully and Bidochka (2005), will provide valuable
sources of phenotypically diverged fungal popula-
tions, which can be thoroughly screened for cor-
related responses in fitness traits under different
environmental conditions; and coupled with
genetic manipulation and biochemical analyses
one can expect to progress in shedding light on
the function of toxic secondary metabolites as
virulence factors.
B. Predator–Prey
Fungi, as an important part of decomposer com-
munities, contain high amounts of nitrogen and
phosphorus, one reason why all fungal structures,
e.g. hyphae and spores, are susceptible to preda-
tion by animals, especially the soil fauna (Ruess
and Lussenhop 2005). Common soil arthropods,
such as mites, collembola, diplura, protura, and
nematode worms, possess specialised mouthparts,
making them excellent fungal feeders (Ruess and
Lussenhop 2005) and some studies demonstrate
changes in mycelial growth and respiration of
various soil fungi in response to collembolan
grazing (Hedlund et al. 1991; Bengtsson et al.
1993; Kampichler et al. 2004; Harold et al. 2005),
thus providing evidence of fitness loss in fungi
due to fungivory (Guevara et al. 2000; Tordoff
et al. 2006). In contrast, fungivorous arthropods
have been selected for abilities that allow them
to discriminate between low- and high-quality
fungal diets, which positively correlates with
important fitness parameters (e.g. Scheu and Sim-
merling 2004; Jørgensen et al. 2005) and fungi-
vores may base their food choice decisions on
volatiles emitted by the fungi (Bengtsson et al.
1991; Hedlund et al. 1995). Such behavioural
plasticity in fungivores has repeatedly been linked
to the toxicity of the fungal diet (Shaw 1988; Ruess
142
et al. 2000) and frequently observed dietary mix-
ing strategies in soil arthropods may be adaptive
in terms of diluting the amount of fungal toxic
compounds (Freeland and Janzen 1974; Singer
et al. 2002). From the fungal perspective, optimal
foraging strategies in fungivores (i.e. avoidance of
toxic fungi) may provide relief from fungivory
and hence a fitness benefit to the fungi. For this
reason, chemical defence based on toxic second-
ary metabolite production is a widespread and
often-cited hypothesis to explain the existence of
insecticidal compounds in fungi. Although, as for
the proposed role of secondary metabolites in
pathogenic fungi, a causal relationship between
toxin production and reduced fitness in the fun-
gus’ predator coupled with a benefit to the fungus
remains to be demonstrated. A recent investiga-
tion used a transgenic strain of A. nidulans that
lacked the expression of laeA and hence the ability
to synthesise several known and unknown sec-
ondary metabolites (Bok and Keller 2004). As de-
scribed above, the DlaeA mutant has been
demonstrated to be unable to produce various
secondary metabolites including penicillin, hy-
1.0
wild type A. nidulans
ΔlaeA A. nidulans
Δ Proportion springtails +/- SE
0.8
0.6
0.4
0.2
0.0
1 2
Time [days]
Fig. 7.6. Effect of chemical deficient mutant DlaeA As-
pergillus nidulans on the feeding behaviour of the
springtail Folsomia candida. The left panel shows the
tendency of 20 springtails to form groups on one of two
colonies of the wild type or the DlaeA strain of A.
nidulans over several days. The measurement ‘D propor-
tion springtails’ indicates the degree of group-formation
(values approaching ‘0’ indicate regular distribution be-
tween the colonies whereas ‘1’ indicates that all animals
gathered on one colony; ‘D proportion springtails’ was
calculated by dividing the difference between the num-
ber of animals on colony one and the number on colony
two by the total number of animals on both fungal
Marko Rohlfs et al.
phal pigments, and the mycotoxin sterigmatocys-
tin (Bok and Keller 2004). In an ecological
experiment, fungivorous soil-dwelling springtails
(Folsomia candida) displayed a distinct prefer-
ence for this chemical-deficient mutant of A. nidu-
lans and the transgenic fungus, compared with a
wild-type strain capable of producing the entire
arsenal of secondary chemicals, suffered from a
significant biomass loss (Rohlfs et al. 2007;
Fig. 7.6). Moreover, as can be seen from Fig. 7.6,
groups of springtails displayed a significantly
stronger aggregation (group formation on one of
two fungal colonies offered) on the wild type than
on the chemical-deficient A. nidulans strain. In
analogy to plant–herbivore systems (Fordyce
2003), this behaviour may reflect an adaptive
strategy for the arthropods to manipulate fungal
suitability as a food source. Because the tendency
to form a large group of foraging individuals on
one fungal diet was less strong when springtails
were offered two colonies of the DlaeA mutant,
chemical diversity as regulated by laeA appears to
play an important role. The behaviour of the
springtails in this set-up clearly demonstrate that
3 4 5
colonies). There was a significant effect of the fungal
diet on the degree of group formation, but no influence
of time (repeated measurement ANOVA; fungal strain: F
= 44.97, d.f. = 1, P < 0.0001, time: F = 2.13, d.f. = 4, P =
0.0791, fungal strain  time: F = 0.86, d.f. = 4, P =
0.4877). The right panel illustrates the effect of springtail
feeding on fungal development (A DlaeA, B wild-type A.
nidulans) and the influence of the fungal strain on the
distribution of the arthropods between the two colonies
(A springtails feed on both colonies; B springtails feed
only on the left colony; arrows point at some individual
springtails). See Rohlfs et al. (2007) for methodological
details
 Evolutionary and Ecological Interactions of Mould and Insects
they base their food choice decisions on cues
related to the ability to synthesise secondary
metabolites and, as suggested by Rohlfs et al.
(2007), the secondary metabolites themselves.
These observations provide genetic support for
the hypothesis that fungi may contain secondary
metabolites as an anti-predator adaptation and
illustrate how the lack of fungal chemicals is
driving the food choice behaviour of fungivorous
arthropods; yet, since laeA globally regulates sec-
ondary metabolism in Aspergillus, the key fungal
compounds involved in deterring fungivores and
improving chemical protection in this ecological
interaction are still unknown.
C. Interspecific Competition
Although micro-organisms, including filamentous
fungi, are considered to function as important
decomposers and recyclers in almost all ecosys-
tems, they can behave like classic consumers of
critical energy and nutrient resources. They may
be engaged in competition with other microbes
but may also compete against representatives
Rohlfs Frontiers in Zoology 2005
2:2 doi:10.1186/1742-9994-2-2
143
from higher trophic levels, i.e. animals (Janzen
1977; Cipollini and Stiles 1993; Crist and Friese
1993; Burkepile et al. 2006). Indeed, competition
has been documented to occur between various
mould species and saprophagous insect larvae,
such as drosophilid flies (Atkinson 1981; Hodge
1996; Hodge et al. 1996; Hodge and Arthur 1997;
Rohlfs 2005a, b, 2008; Rohlfs and Hoffmeister
2005; Rohlfs et al. 2005). In line with the general
assumption about interspecific competition
(see above), the outcome of Drosophila–fungus
competition strongly depends on priority effects
(i.e. the age of fungal colonies at the time insect
larvae enter a resource patch) and insect density
(Rohlfs 2005b; Rohlfs et al. 2005; Fig. 7.7). More-
over, the negative effect on insect development
varies among fungal species (Fig. 7.8), even with
some fungi causing no mortality in the insects, but
themselves suffering from the presence of the in-
sect larvae (e.g. Penicillium sp.; Rohlfs et al. 2005).
Impairment of rapid expansion of a fungal
colony (or entire suppression of fungal growth)
on a resource patch appears to be due to physical
(and/or chemical?) destruction of tissue in
the mould’s active growth zone by the insect
Fig. 7.7. Aggregation of Dro-
sophila melanogaster larvae on
fungal colonies and its effect on
fungal development. A Several
Drosophila larvae ‘attacking’ a
two-day-old colony of Aspergil-
lus fumigatus. B Effect of ten
Drosophila larvae (lower Petri
dish) on the growth of Aspergil-
lus nidulans compared with an
undisturbed colony (upper Petri
dish). Colonies were four days
old, growing on Drosophila
corn meal/yeast hydolysate me-
dium at 25 C. C Impact of one
(left), five (middle) and ten
(right) Drosophila larvae on the
growth of Aspergillus niger ten
days after inoculation on Dro-
sophila medium
144
1.0
Drosophila larval survival
0.8
0.6
0.4
0.2
0.0
–2
Fig. 7.8. Fungus-specific effects on Drosophila survival
and experimental setup. Left panel Impact of three Asper-
gillus species on the survival of Drosophila larvae to the
adult stage as a function of fungal priority (e.g. ‘0’ denotes
simultaneous transfer of larvae and fungal conidia, ‘1’ and
‘ 1’ fungal or insect ‘head start’ of one day, respectively.
Dashed line indicates insect survival under mould-free
condition. For each treatment N = 20. For clarity, error
bars are omitted. Right panel Aspergillus–Drosophila
larvae (Fig. 7.7). Despite the eventually negative
effect of, e.g. Aspergillus fungi, on an insect feed-
ing substrate, Drosophila larvae display conspicu-
ous aggregation behaviour on fungal colonies
(Rohlfs 2005a; Fig. 7.7). This behavioural response
has been correlated with retarded mould growth, a
relationship that becomes especially pronounced
when the effect of increasing insect density is
taken into account (Fig. 7.7). Importantly, fungal
growth as determined by Drosophila larval density
has been shown to be negatively correlated to
insect survival (Hodge 1996; Rohlfs et al. 2005).
Although at the moment we cannot exclude any
nutritional benefit from feeding on young fungal
tissue to the insects, one interpretation of this
behaviour and its consequences for both the in-
sect and the fungus is that it is part of an animal
defensive response to the harmful mould (Rohlfs
2005a). The clearly active interaction between the
fungi and the animal antagonists indicates a
strong interference rather than exploitation com-
petition. The latter signifies competition without
direct antagonistic contact between competitors.
However, organisms compete by capturing res-
ources faster than their competitors.
Interestingly some fungi, such as A. flavus, are
still able to successfully invade resources and kill a
significant proportion of larvae in which the im-
mature insects have already started feeding
(Fig. 7.8). Since A. flavus and A. fumigatus have
been described as opportunistic pathogens (see
Marko Rohlfs et al.
A. nidulans
A. flavus
A. fumigatus
–1 0 1 2 3 4
Fungal priority [days]
confrontation takes place in 2 ml microtubes filled with
1 ml standard Drosophila corn meal/yeast hydolysate me-
dium and sealed with cotton plug (dental roll). Storing
these miniaturised experimental units in racks allows
preparation of high numbers of simultaneous replicates.
Following a standard protocol, experimental units are
inoculated with 1000 conidiospores in 1 ml Ringer solu-
tion. Subsequently, the experimental setup is incubated at
25 C and a 16-h light cycle
Sect. III.A), Drosophila larval mortality might be
attributable to a pathogenic effect; however, there
is as yet no evidence that these fungi do infect the
insect larvae. Continuous exposure to fungal spores
during larval development did not induce higher
mortality rates than development on a sterile sub-
strate (Fig. 7.9). The interpretation of these results
is based on the current knowledge on how insect
get infected by pathogenic fungi (Clarkson and
Charnley 1996; see also Sect. III.A). We suppose
that infection of the Drosophila larval stage is, if at
all, only possible via attachment of spores to the gut
epithelium, since the physical conditions due to the
foraging and digging behaviour of the larvae in the
feeding substrates appears to be unlikely to lead to
a durable spore–host contact that is long enough to
allow the fungal infection process to occur via the
larval exoskeleton. As indicated by the data shown
in Fig. 7.9, however, also infection via the larval gut
does not seem to occur (for non-pathogenic effects
of injection of A. fumigatus spores into adult flies,
see Chamilos et al. 2008). It seems to be a general
phenomenon that Drosophila is rather insuscepti-
ble to fungal infections during the larval stage, even
if fungi are classified as entomopathogens, such as
B. bassiana (Kraaijeveld et al. 2008).
Such non-pathogenic but competitive interac-
tions between fungi and insects may have far-
reaching consequences for trophic interactions
within the animal communities. For instance, in-
festation of larval insect feeding substrates with
 Evolutionary and Ecological Interactions of Mould and Insects 145

1.0
1.0

Survival to adult stage +/-SE

Sterigmatocystin
O OH
Survival to pupal stage +SE
0.8 0.8 O

O O O
CH3

0.6 0.6

0.4
0.4

0.2
0.2
0.0
0 2 4 6 8
0.0 µg per 1ml fly medium
l

us

s
tro

an

vu
at
n

Fig. 7.10. Effect of sterigmatocystin at varying concentra-

fla
ul

ig
co

id

A.
n

fu

tions on the survival of Drosophila melanogaster larvae to

A.

A.

the adult stage

Fig. 7.9. Survival to the pupal stage of Drosophila mela-
nogaster larvae as a function of continuous exposure to
spore of various Aspergillus species. For a period of one
week, larvae were transferred each day to fresh medium
containing new conidiospores ( one million). These data
illustrate that Drosophila larvae are quite resistant to As-
pergillus via the common way of infection, as described in
Sect. III.A, supporting the proposed competitive interac-
tion between mould and saprophagous insects
A. niger had a strong negative effect on Drosophila
larvae carrying the eggs/larvae of a parasitic wasp
(Rohlfs 2008). When parasitised Drosophila larvae
were forced to develop on fungus-infested sub-
strates, they suffered (depending on larval densi-
ty; see above) from higher mortality than healthy
larvae. This could negatively influence the popu-
lation growth of parasitic wasps. Moreover, forag-
ing wasps abandoned searching for host larvae
significantly earlier on mould-infested patches
than they did on mould-free substrate (Rohlfs
2008), implying that the fungus allows the Dro-
sophila larvae to feed in a refuge which protects
the larvae from parasite attacks. Interestingly, be-
cause A. niger had significantly enhanced coni-
diospore production when the fungus was
confronted not with unparasitised but with para-
sitised Drosophila larvae (Rohlfs 2008), antagonis-
tic processes within the insect communities have
the potential to control reproduction of the fungal
competitor and hence microbial community
structure.
Besides these immediate ecological effects of
saprophytic mould on saprophagous insect com-
munities, fungi can be expected to influence the
evolution of Drosophila behaviour and life history
strategies. In the view of the severe direct effects
on insect development, the insects are likely to be
selected to resist the fungal competitor. Yet by
using the isofemale line technique Rohlfs (2006)
demonstrated genetic variation in the ability of
Drosophila to successfully develop in the presence
of A. niger. Heritable variation in the survival of
insect larvae ranged from less than 20% to almost
100%, which reveals the potential of fungi to select
for improved insect development when con-
fronted with the fungal competitor. From an adap-
tationist’s perspective, the fact that insect
populations display variation in the ability to re-
sist fungal competitors suggests that the animals
have to ‘pay’ some cost for the increased resis-
tance. Indeed replicated fly populations selected
for resistance to A. nidulans during the larval
stage show a reduced ability to withstand abiotic
stress and to fend off parasites (Rohlfs, Trienens,
Wölfle, unpublished data).
Given the diverse fatal but non-pathogenic
effects of mould on saprophagous insects, fungal
secondary metabolites are prime candidates for
explaining the ecological and evolutionary impact
of fungi on Drosophila and vice versa. First evi-
dence of a critical role of toxic secondary metabo-
lites comes from various pharmacological tests.
Prominent mycotoxins, such as aflatoxin, patulin,
kojic acid (Reiss 1975; Dobias et al. 1977; Chinnici
et al. 1979; Rohlfs 2008), and sterigmatocystin
(Fig. 7.10) are toxic to Drosophila during larval
development. Second, insect mortality drastically
increases with increasing age of fungal colonies
prior to larval settlement in a resource patch
146 Marko Rohlfs et al.
(Fig. 7.8). These strong priority effects (see above)
might be attributable to the positive relationship
between fungal development and toxic secondary
metabolite production (Calvo et al. 2002). Howev-
er, as for the pharmacological studies this inter-
pretation is based on observations on correlative
rather than causal relationships. For instance,
pharmacological studies are confounded by the
altered ecological context and chemical milieu in
which both insect and fungal resistance traits may
evolve: fungi critically modify the resource (e.g.
pH), secrete numerous extracellular enzymes
(hydrolases), and occupy space on a limited re-
source patch that cannot be exploited by the
insects. Moreover, fungi such as Aspergillus sp.
are well known for producing many different sec-
ondary metabolites, which may lead to unforesee-
able synergistic effects (Creppy et al. 2004) that
may drive both the efficiency of the chemical
defence and the evolution of resistance in compet-
ing insects. And the most exciting question in this
context is whether fungi are able to adjust their
chemical armoury to the corresponding ecological
challenge since biosynthesis of e.g. mycotoxins
can be considered costly for the organism
(Shwab et al. 2007), or if temporal patterns in
secondary metabolite production represent an in-
flexible constitutive defence system that is main-
tained even in the absence of animal antagonists.
Even though fungi have been shown to possess the
mechanistic prerequisites for regulating their
chemical diversity, it has not yet been demon-
strated whether: (1) this machinery is capable of
responding to e.g. competing fungi or predatory
arthropods and (2) such proposed responses are
adaptive in terms of reducing the opponent’s im-
pact on fungal development. From a fungal mo-
lecular perspective, the major goal is to
understand whether genes and gene clusters
involved in secondary metabolite biosynthesis
function as ‘environmental response genes’ that
exhibit condition and temporal specific expres-
sion (c.f. Berenbaum 2002).
IV. Melding Ecology and Molecular
Biology
Given the widespread ability to produce some of
the most potent toxins in the living world, second-
ary metabolite biosynthesis can be viewed as a
successful evolutionary outcome. Yet the ecologi-
cal and evolutionary mechanisms that influence
or underlie this success still remain elusive. To
resolve the interplay between fungal secondary
metabolite genes and the ecological process in
which secondary metabolite production is pro-
posed to be involved, we suggest to apply the
emerging field of evolutionary and ecological
functional genomics (Feder and Mitchell-Olds
2003), which requires the simultaneous use of
molecular, organismal, and ecological approaches
(Jackson et al. 2002; Thomas and Klaper 2004).
The application of this perspective provides an
integrative understanding of the interrelationship
of genes, the associated pathways, and the
corresponding phenotypes involved in the ecolog-
ical process. We believe we have identified the
Aspergillus–Drosophila system as being suitable
to reveal the ecological and evolutionary impor-
tance of fungal secondary metabolites, since both
organisms fulfil the basic prerequisite of
completely sequenced genomes for being models
in genomics (Adams et al. 2000; Galagan et al.
2005). Also the rapid advances in developing
high-throughput microarrays enable global gene
expression analyses in both fungi and insects
(Gupta and Oliver 2003; Andersen et al. 2008)
and, from an ecological point of view, this offers
the unique opportunity to study simultaneous
changes in gene expression in both competitors.
A preliminary genome-wide screening for changes
in transcriptional activity in D. melanogaster
larvae confronted with A. nidulans indicates the
up-regulation of several genes, e.g. those involved
in antimicrobial immune defence and insecticide
resistance (Trienens and Rohlfs, unpublished
data). Likewise quantitative RT-PCR of RNA
from A. nidulans confronted with D. melanogaster
larvae indicates the up-regulation of secondary
metabolite regulator genes (Fohgrub and Kemp-
ken, unpublished data). To our current knowl-
edge, the possibilities of using expression
profiling techniques for Aspergillus have not yet
been applied in the context of species interaction.
But for differentially expressed genes in A. fumi-
gatus in response to variation in vertebrate im-
mune response, see Sugui et al. (2008); and for a
recent review on microarray studies in filamen-
tous fungi, see Breakspear and Momany (2007).
The second reason why organisms can become
models in genomics and hence ecological geno-
mics is the possibility of genetic manipulation.
 Evolutionary and Ecological Interactions of Mould and Insects
Being able to knock-out, down-regulate, or over-
express candidate genes against a genetic back-
ground that is the same as that of the wild type
will be necessary to validate the causal relationship
between e.g. expression of secondary metabolites
genes in fungi and Drosophila larval development
(van Straalen and Roelofs 2006; Kammenga et al.
2007). Transposon-based mutagenesis may be use-
ful as an additional tool (Kempken 1999; Pöggeler
and Kempken 2004; Braumann et al. 2007). Based
on a priori selection of the candidate gene laeA,
Rohlfs et al. (2007) have already provided evidence
of the usefulness of this approach in a fungus–
fungivore system; however, this study did not dis-
tinguish between the causal agent(s) driving fun-
givore behaviour and unimportant secondary
metabolite pathways, since laeA is a regulator of
many secondary metabolites (see above). Critical
advances may be achieved by manipulating gene
expression based on differentially expressed fun-
gal genes as stimulated by insect attack, and testing
whether transgenic fungi confirm the hypothe-
sised role of genes involved in secondary metabo-
lite biosynthesis in the corresponding ecological
setting. A comprehensive molecular toolbox is
available to achieve this goal in Aspergillus (Gold-
man and Osmani 2008). The same experimental
strategy can be applied to the insects. By using the
binary GAL4/UAS system to generate even tissue-
specific post-transcriptional silencing of candidate
genes in Drosophila (Dietzl et al. 2007) will provide
unparalleled insights into the mechanistic basis of
the role fungal defences against competing insects
and the animals’ counter-adaptations. Moreover,
coupling this molecular approach with experimen-
tal evolution as described above will enable us to
thoroughly study the possible role of co-evolution-
ary processes in this ecological interaction. How-
ever, such studies are currently still under
development. As pointed out by Jackson et al.
(2002), integrating molecular and ecological re-
search calls for interdisciplinary approaches and
hence intensive collaboration between molecular
biologists and ecologists.
V. Conclusions
Fungus–insect relationships range across symbio-
sis, mutualism, and antagonism. Here we concen-
trate on various antagonistic interactions between
147
typical mould species and various insect groups.
Independently of whether fungi and insects are
engaged in parasitism, predation, or competition,
the production of secondary metabolites has been
proposed to incur a key function within fungi in
attacking insects or defending against them. Many
studies provide correlative or pharmacological ev-
idence of such a role of mycotoxins. Future molec-
ular genomic approaches in combination with
experimental evolutionary ecology will forge a
mechanistic basis for fungus–insect interactions
that will be able to answer fundamental questions
regarding the biological significance of fungal sec-
ondary metabolism. We are still at the stage of
having only vague arguments for fungal toxins
being part of a chemical defence system against
insects; but the use of carefully designed transgen-
ic fungi coupled with biochemical analyses will
shed light on such a function of possibly still un-
known secondary metabolites. An evolutionary
ecological genomic approach will also help us
proving that secondary metabolite production is
indeed costly to fungi and that the biosynthesis of
e.g. mycotoxins is related to the ecological context,
such as insect attacks. It remains an open question
whether the signalling of changes in environmen-
tal condition in fungi, as controlled by oxylipins
(Tsitsigiannis and Keller 2007), have its ecological
analogy in plant–insect systems (Kessler et al.
2004). To our knowledge the molecular regulatory
machinery of secondary metabolite synthesis and
its proposed function in controlling the allocation
of limited resources to costly insecticidal mole-
cules in relation to the threat of being attacked by
natural enemies have yet to be implemented. Hav-
ing a good ecological model system at hand (e.g.,
Aspergillus–Drosophila) will finally allow us to draw
an evolutionary line between the organisation of
fungal defence against natural enemies and viru-
lence in invertebrate and vertebrate hosts.
Acknowledgement. The laboratory work of F.K. and M.R.
is funded by the German Research Council (DFG).
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8 Endophytic Fungi, Occurrence and Metabolites

DANIELA WEBER1

CONTENTS C. Metabolites from Endophytic

Penicillium Species . . . . . . . . . . . . . . . . . . . . . . . . . . 170
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 1. Metabolites from Penicillium sp.
II. The Ecological Relevance from Aegiceras corniculatum L. . . . . . . . 170
of Endophytic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 2. Metabolites from Penicillium
III. Metabolites Isolated from Host Plants chrysogenum from Cistanche
and Their Endophytic Fungi . . . . . . . . . . . . . . . . . . . 156 deserticola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
A. Taxol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 3. Metabolites from Penicillium spp.
B. Camptothecin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 from Prumnopitys andina . . . . . . . . . . . . . 171
C. Ergot Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 4. Penicidones from Penicillium sp.
D. Mycotoxins in Baccharis Species . . . . . . . . . . 157 from Quercus variabilis . . . . . . . . . . . . . . . . 174
E. Hypericin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 D. Metabolites from Endophytic Alternaria
F. Podophyllotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
IV. Metabolites from Endophytic Fungi . . . . . . . . . . . 159 1. Metabolites from Alternaria sp.
A. Metabolites from Endophytic from Polygonum senegalense . . . . . . . . . . 174
Xylariaceous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . 159 2. Metabolites from Alternaria spp.
1. 7-Amino-4-Methylcoumarin from from Vinca minor and
Xylaria sp. of Ginkgo biloba . . . . . . . . . . . 159 Euonymus europaeus . . . . . . . . . . . . . . . . . . . 175
2. Metabolites from Xylaria sp. from E. Xanthones from Emericella variecolor
Sandoricum koetjape . . . . . . . . . . . . . . . . . . . 159 from Croton oblongifolius . . . . . . . . . . . . . . . . . . 175
3. Sesquiterpenoids from a F. Cyclopentenons from Dothideomycete
Xylariaceous Fungus . . . . . . . . . . . . . . . . . . . 163 sp. from Leea rubra . . . . . . . . . . . . . . . . . . . . . . . . . 175
4. Metabolites from Xylaria sp. . . . . . . . . . . 163 G. Metabolites from Endophytic
B. Metabolites from Endophytic Phomopsis Chaetomium Species . . . . . . . . . . . . . . . . . . . . . . . . 176
or Diaporthe Species . . . . . . . . . . . . . . . . . . . . . . . . 164 1. Metabolites from Chaetomium sp.
1. Lactones from Phomopsis sp. from from Nerium oleander L. . . . . . . . . . . . . . . 176
Azadirachata indica . . . . . . . . . . . . . . . . . . . . 164 2. Cytochalasan Alkaloids from
2. Metabolites from Diaporthe sp. from Chaetomium globosum from
Camellia sinensis L. . . . . . . . . . . . . . . . . . . . . 165 Imperata cylindrica . . . . . . . . . . . . . . . . . . . . . 177
3. Metabolites from Phomopsis sp. from H. Metabolites from Endophytic
Camptotheca acuminata . . . . . . . . . . . . . . . 166 Pestalotiopsis Species . . . . . . . . . . . . . . . . . . . . . . . 178
4. Sesquiterpenoids from Phomopsis 1. Isopestacin from Pestalotiopsis
cassiae from Cassia spectabilis . . . . . . . . 166 microspora from Terminalia
5. Metabolites from Phomopsis spp. morobensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
from Erythrina crista-galli . . . . . . . . . . . . . 166 2. Sesquiterpenes from Pestalotiopsis
6. Metabolites from Phomopsis sp. sp. from Pinus taeda . . . . . . . . . . . . . . . . . . . 178
from Eupatorium arnottianum . . . . . . . . 169 3. Metabolites from Pestalotiopsis
7. Cytosporone D from Phomopsis sp. foedan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
of Phytolacca dioica . . . . . . . . . . . . . . . . . . . . 170 4. Metabolites from Pestalotiopsis
8. Xanthone Dimers from Phomopsis theae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
sp. from Tectona grandis L. . . . . . . . . . . . 170 J. Metabolites from Phyllosticta spinarum
9. Dicerandrols from Phomopsis from Platycladus orientalis . . . . . . . . . . . . . . . . . 180
longicolla from the Mint K. Metabolites from Endophytic
Dicerandra frutescens . . . . . . . . . . . . . . . . . . 170 Fusarium Species . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
1. CR377 from Fusarium sp. from
Selaginella pallescens . . . . . . . . . . . . . . . . . . . 181
1
2. Lipopeptides from Fusarium sp.
Institute of Biotechnology and Drug Research, Erwin-Schröd- from Maackia chinensis . . . . . . . . . . . . . . . . 181
inger-Strasse 56, 67663, Kaiserslautern, Germany; present address: L. Epichlicin from Epichloe typhina from
MIP International Pharma Research GmbH, Mühlstrasse 50, Phleum pretense L. . . . . . . . . . . . . . . . . . . . . . . . . . . 181
66386 St. Ingbert, Germany; e-mail: weber-daniela@gmx.de

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
154 Daniela Weber

M. Metabolite from Colletotrichum dematium and xylem vessels within the plant, to more or less
from Pteromischum sp. . . . . . . . . . . . . . . . . . . . . 181 superficial colonization of peripheral, often dying
N. Hormonemate from Hormonema or dead tissues such as bark layers on plants with
dematioides from Pinus sp. . . . . . . . . . . . . . . . . 182 secondary growth (Petrini 1996). Ascomycetes,
O. Brefeldin A from Phoma medicaginis
from Medicago lupulina . . . . . . . . . . . . . . . . . . . . 183
Basidiomycetes, Deuteromycetes, and Oomycetes
P. Phaeosphaerides from an Endophytic were isolated as endophytes (Sinclair and Cerkaus-
Fungus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 kas 1996); of these the Ascomycetes and imperfect
Q. Metabolites from Nodulisporium fungi constitute the largest group.
sp. from Junipercus cedre . . . . . . . . . . . . . . . . . . 183 In some cases this fungus–plant interaction
R. Metabolites from Ascochyta sp. from
Meliotus dentatus . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
has mutualistic features. Enhanced rate of growth
S. Metabolites from Endophytic Fungi (Bradshaw 1959; Kackley et al. 1990; Hill et al.
from Garcinia Species . . . . . . . . . . . . . . . . . . . . . . . 184 1991), resistance and toxicity against herbivores
T. Metabolites from Endophytic Fungi (Wallner et al. 1983; Read and Camp 1986), deter-
from Artemisia species . . . . . . . . . . . . . . . . . . . . . . 187 rence of insects (Clay 1988b), resistance against
U. Metabolites from Endophytes from
Schinus molle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
nematodes (Kimmons et al. 1990; West et al. 1990)
V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 and pathogens (White and Cole 1985; Yshihara
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 et al. 1985), and enhanced drought tolerance
(Read and Camp 1986; Arechavaleta et al. 1989;
West et al. 1993) are observed as advantages to the
host plants. The benefits for the fungal symbionts
can be greater access to nutrients, protection
I. Introduction against desiccation, insects, and parasitic fungi,
and competition from other microbes (White
Symbiosis is defined in the original sense as the et al. 2000). During the coevolution, endophytic
living together of different organisms (de Bary microbes improved the resistance of the host
1879). Perhaps two-thirds of the known species of plants to adverse conditions by secreting bioactive
fungi form intimate relationships with other living secondary metabolites (Ge et al. 2008).
organisms in parasitic, commensalistic, or mutual- Although the first discovery of an endophytic
istic symbiosis (Pirozynski and Hawksworth 1988). fungus was in 1902 by Freeman, little attention
There exist different fungus–plant interactions, was paid to this group of fungi until the recent
which range from mycorrhiza to plant pathogens. realization of their ecological relevance and their
Arbuscular mycorrhizal fungi are ancient Zygomy- potential as source for new bioactive metabolites
cetes and are associated with the roots of about 80% (Gunatilaka 2006).
of plant species (Bonfante and Perotto 1995). This
fact supports the hypothesis of Pirozynski und Mal-
loch (1975) that the colonization of land and the II. The Ecological Relevance
evolution of plants were possible only through the of Endophytic Fungi
establishment of symbiotic association of a semi-
aquatic ancestral alga and an aquatic fungus. Two ecological groups of endophytes can be dis-
Of the estimated 1.5
106 fungal species on tinguished: grass endophytes and endophytes of
earth (Hawksworth 2001) about 1.30
106 species woody plants. Endophytic fungi were found in all
are estimated to live as endophytes in plants (Drey- woody plants examined for endophytes (Saikko-
fuss and Chapela 1994). So most of the fungi are nen et al. 1998). Despite the great diversity and
endophytes. They are ubiquitous, and probably all abundancy of endophytes in woody plants, these
vascular and non-vascular terrestrial plants harbor endophytes and their interaction with their host
them (Weber and Anke 2006). The term ‘‘endo- plants receive less attention than those of grass
phyte’’ includes all organisms that, at some time endophytes (Saikkonen et al. 1998). The produc-
of their life cycle, live symptomlessly within plant tion of alkaloids in infected grasses (in planta) can
tissue (Petrini 1996). This definition also includes cause intoxication of grazing livestock. Because of
pathogens with extended latency periods. Endo- this economic importance, endophytes of poa-
phyte association may range from intimate contact ceous grasses are the best-examined (Weber and
where the fungus inhabits the intercellular spaces Anke 2006).
 Endophytic Fungi, Occurrence and Metabolites
In the family Clavicipitaceae there is a devel-
opment from parasitic grass endophytes like Cla-
viceps to mutualistic species like Acremonium
(anamorphic form of Epichloë; Clay 1988a). Spe-
cies of the genus Claviceps cause local infections
of the inflorescences of grasses. Sclerotia develop
at the site of infection instead of seeds of the host
plant. The local infection evolved to a systemic
infection, which can be found in Acremonium
species (Clay 1988a). Reduction of sexual repro-
duction can be observed in this group of obligate
endophytic fungi (Saikkonen et al. 1998). The
asexual or anamorphic form is transmitted verti-
cally by the growth of fungal hyphae into the host
plant’s seeds (Arnold et al. 2003). Vogl already
discovered the mycelium of an endophytic fungus
in seeds of Lolium temulentum in 1898. Infection
of the inflorescenses by parasitic species results in
sexual sterilization of the host plant. This is in
contrast to the loss of sexual reproduction of
endophytes in mutualistic symbiosis. In these
cases propagation of the fungus is ensured by
viable seeds of the host plant.
The endophytic fungi of woody plants show a
high diversity and belong to different taxonomic
groups. These fungal endophytes are horizontally
transmitted via spores and are not known to
grow into seeds and systemically infecting the
plant following seed germination (Wilson 2000).
The infections are often highly localized within
leaves, petioles, bark, or stems (Saikkonen et al.
1998).
Many endophytes of woody plants are closely
related to pathogenic species. Freeman and Rodri-
guez (1993) were able to show that the mutation at
a single genetic locus can change an isolate of
Colletotrichum magna from a pathogen to a non-
pathogenic endophytic mutualist. The wild-type
fungus and the mutant of the species (path 1)
were capable to infect the host plant and systemic
growth was observed. The host plants infected by
path 1 produced no visible effect on any plant and
were protected from the wild-type fungus. Free-
man and Rodriguez (1993) assumed that a portion
of the host defense system is activated by path 1 so
that, when exposed to the wild-type fungus, there
was no delay in the defense response.
In contrast to the fungi of the Clavicipitaceae
the endophytes of woody plants are not intensive-
ly investigated. One exception is the family of the
Xylariaceae, which attracts attention because of
its fruiting bodies and its host specificity. Most
155
xylariaceous species are collected from living or
dead angiospermous plants (Rogers 1979); most
members of the family are wood inhabitants but
representatives are also found in litter, soil, dung,
and associated with insects (Whalley 1996). The
greatest diversity of xylariaceous fungi can be
found in the tropics (Petrini et al. 1995). Whalley
(1996) distinguishes three groups: saprophytes,
phytopathogens, and endophytes. Endophytic
species, e.g. Hypoxylon and related genera, can
colonize the xylem of host plants (Stone et al.
2000). Other xylem-colonizing endophytes have
been identified as species of the Diaporthales (e.
g. Phragmoporthe, Amphiporthe, Phomopsis),
Hypocreales (Nectria spp.), and a few Basidiomy-
cetes (e.g. Coniophora; Stone et al. 2000).
Water deficiency and drought in the host
plant can cause a change in the lifecycle of endo-
phytic species. Many plant-pathogenic species of
the Xylariaceae have typical characteristics of
endophytes; they live as ‘‘latent invaders’’. Water
stress triggers the switch to the pathogenic phase
of the fungus (Whalley 1996). A switch from the
latent state to the saprophytic stage is observed in
Hypoxylon fragiforme when the colonized host
tissue dies. The death of the tissue is accompanied
by loss of water, which triggers the switch to the
saprophytic stage of fungus (Weber and Anke
2006).
Some of the xylariaceous fungi have devel-
oped host specificity, e.g. Hypoxylon fragiforme
is consistently isolated from healthy beech trees
in Europe and H. mammatum is isolated from
aspen trees in the northeast of the United States
(Chapela and Boddy 1988; Chapela 1989). The
wide distribution of the Xylariaceae, as endo-
phytes and as saprophytes and their ability to
produce bioactive compounds (cytochalasins,
melleins, xylarin) suggest that these fungi have
an important ecological role (Petrini et al. 1995).
Compounds from the bark of beech trees can
trigger the eclosion of Hypoxylon fragiforme
ascospores, which is a precondition for germina-
tion of the spores (Chapela et al. 1993). This can
partly explain the host specificity of H. fragiforme.
The eclosion of the spores can be observed 10 min
after the addition of the beech extract (Webster
and Weber 2004). Two substances have been iden-
tified which trigger the release of the spores at
micromolar concentrations: the two monolignol
glucosides Z-syringin and Z-isoconiferin (Fig. 8.1;
Chapela et al. 1991).
156 Daniela Weber

O OH OH
HO HO
OH OH
O O O
O

OH OH
O

OH OH

Z-Syringin Z-Isoconiferin

O
O O OH
H

N O
O N
O NH O

O O
OH O
OH O O
O

O
Taxol Camptothecin

Fig. 8.1. Structures of Z-syringyn, Z-isoconiferin, taxol, and camptothecin

III. Metabolites Isolated from Host
Plants and Their Endophytic Fungi
A. Taxol
Endophytes are a rich source for bioactive meta-
bolites and therefore have opened an interesting
field of natural product research (Strobel et al.
2004; Ding et al. 2006). A well-known example is
the antitumor agent taxol (paclitaxel) which binds
to b-tubulin and promotes the stabilization of
microtubules by inhibiting depolymerization of
microtubules to soluble tubulin (Jennewein and
Croteau 2001). Especially, the fast proliferation of
tumor cells is inhibited by this mode of action.
The original targeted diseases for taxol were ovar-
ian and breast cancer, but now it is also used for
the treatment of other human tumors (Strobel
et al. 2004). The diterpenoid taxol (Fig. 8.1) was
first isolated from the bark of the pacific yew
(Taxus brevifolia; Wani et al. 1971). All Taxus
species produce taxol, or related taxane diterpe-
noids (taxanoids) and over 350 different taxoid
structures are already known (Jennewein and Cro-
teau 2001; Strobel 2003). In 1993 Stierle and cow-
orkers reported that the endophyte Taxomyces
andreanae, which was isolated from the inner
bark of T. brevifolia, produced the antitumor
agent taxol. Since then, a variety of endophytic
fungi belonging to different categories isolated
from Taxus species have been reported to be ca-
pable of producing taxol and/or taxane derivates
in culture (Tan and Zou 2001).
Since pure cultures of endophytic fungi as well
as cells of Taxus species accumulate taxol, both
must have the genes for taxol synthesis in their
genome. It seems unlikely that the biosynthesis of
an elaborate molecule like taxol developed repeat-
edly during evolution (Kucht et al. 2004). So it is
assumed that a horizontal (lateral) gene transfer
occurred (Kucht et al. 2004). Walker and Croteau
(2001) isolated the genes specific to taxol biosyn-
thesis and noticed that the cDNA sequence of the
taxadiene synthase includes a N-terminal target-
ing sequence for localization and processing in the
plastids. This fact supports the hypothesis that
this gene is plant-derived rather than a fungal
product (Cassady et al. 2004). Ge et al. (2008)
assume that, during the long co-evolution of
endophytes and their host plants, endophytes
have adapted themselves to their microenviron-
ments by genetic variation, including uptake of
 Endophytic Fungi, Occurrence and Metabolites
some plant DNA into their genomes. This could
lead to in the isolation of microbial metabolites
originally known as phytochemicals.
The ecologic relevance of taxol and its synthesis
can be deduced from its antifungal properties.
Oomycetes like Phytophthora, Pythium, and Apha-
nomyces, which can cause diseases in plants, are
extremely sensitive to taxol (Strobel 2003). So it is
almost impossible to find Taxus species that show
any infections caused by any of these Oomycetes
(Strobel 2003). Additionally, the production of
taxol in a T. chinensis cell suspension culture was
stimulated by addition of a fungal homogenate
(Wang et al. 2001). Aspergillus niger, an endophytic
fungus isolated from the inner bark of a T. chinensis
tree, was used as an elicitor to stimulate the taxol
production. Taxol yield was increased to about
seven times the amount obtained in the non-
elicited culture (Wang et al. 2001). This supports
the assumption that taxol is part of the plant
defense against fungi.
B. Camptothecin
Another important antitumor agent is the penta-
cyclic quinoline alkaloid camptothecin (Fig. 8.1).
Camptothecin, first isolated from Camptotheca
acuminata (Wall et al. 1966), has antineoplastic
properties by inhibition of topoisomerases I.
These enzymes relax DNA torsional strain gener-
ated during replication, transcription, recombina-
tion, repair, and chromosome condensation (Meng
et al. 2003). Analogues of camptothecin are used to
treat ovarian, colorectal, and small-cell lung can-
cers, and several second-generation analogues are
in clinical trials (Oberlies and Kroll 2004).
The quinoline alkaloid was also isolated from
the Indian tree Nothapodytes foetida (formely
Mappia foetida; Govindachari et al. 1974). The
compound is available only in low concentrations
in the root of N. foetida, which requires uprooting
of rare and 50- to 75-year-old trees (Puri et al.
2005). In search of alternatives the endophytic
fungi of the tree were investigated and a fungus
producing camptothecin under culture conditions
was identified (Puri et al. 2005). Molecular analy-
sis of the fungus revealed similarity to Entropho-
sphora infrequens and other related taxa, e.g.
Rhizopus oryzae. This was the first report of a
camptothecin-producing microorganism.
157
C. Ergot Alkaloids
Poisoning by the alkaloids of Claviceps species has
been an important problem in human history
(Schardl et al. 2004). These pathogenic fungi infect
grain, and especially rye is very susceptible because
of its open-pollinated grain (Schardl et al. 2004).
After the infection of the host floret the fungus
forms its sclerotium, commonly called an ergot
(Schardl et al. 2006). Sclerotia are a rich source of
secondary metabolites, e.g. ergot alkaloids like er-
gotamine (Parker and Scott 2004). During the Mid-
dle Ages ergot contamination of rye flour caused
mass ergot poisoning (ergotism). The human intox-
ication acquired by eating cereals infected with
ergot sclerotia, usually in the form of bread made
from contaminated flour, is called ergotism or St.
Anthony’s fire (Bennett and Klich 2003). Ergota-
mine (Fig. 8.2) and its derivates share structural
similarities with the adrenergic, dopaminergic, and
serotonergic neurotransmitters and therefore have
wide-ranging effects on the physiological processes
that they mediate (Silberstein and McCrory 2003).
Ergot was used in folk medicine as an abortifacient
and a drug to accelerate uterine contractions
(Bennett and Klich 2003). Today ergotamine and
dihydroergotamine are used for the treatment
of migraine (Aktories et al. 2005). Other ergot deri-
vatives are used as prolactin inhibitors, in the
treatment of Parkinsonism and in cases of cerebro-
vascular insufficiency (Bennett and Klich 2003).
D. Mycotoxins in Baccharis Species
In some cases a direct contribution of the endo-
phytic fungi to the metabolites of the host plant was
observed. The shrubs Baccharis coridifolia and B.
artemisioides, which are widespread in Argentinia,
cause toxic effects in livestock, particularly during
the flowering season (Rizzo et al. 1997). Macrocy-
clic thrichothecenes, roridins and verrucarins,
are the reason for these toxic effects of healthy
B. coridifolia plants (Busam and Habermehl
1982). These metabolites are mycotoxines (e.g.
miotoxin; Fig. 8.2) with cytotoxic properties,
formerly known from different fungi, particularly
of the genus Myrothecium. The isolation of
endophytic fungi from B. coridifolia resulted in
the discovery of a new species, Ceratopycnidium
baccharidicola. One-third of C. baccharidicola
from Argentinian plants produced roridin and
158 Daniela Weber

H H
O
C6H5
O
H O O
O H
N N
N O
H
N H H
O O O O
OH

H
O
NH HO

OH

Ergotamine MiotoxinA

OH O OH OH

O
O
O
OH
O
OH

O O

OH O OH O

Hypericin Podophyllotoxin

Fig. 8.2. Structures of ergotamine, miotoxin A, hypericin, and podophyllotoxin

verrucarin toxins in pure culture (Rizzo et al. 1997).
It can be assumed that the toxins are produced by
the fungi in their host plants, accumulate in the
plant tissue, and may protect both the plant and
the fungus against herbivores.
E. Hypericin
Hypericum perforatum (St. John’s Wort) has been
used for centuries in the treatment of burns,
bruises, swelling, inflammation, and anxiety, as
well as bacterial and viral infections (Hashida
et al. 2008). Traditional indications and uses
include enhancement of wound healing, anti-
inflammatory, and analgesic activities (Miller
1998a). The medicinal plant H. perforatum is com-
monly used for the treatment of mood disorders,
since its effectiveness in the therapy for mild to
moderate depression was claimed to have smaller
side effects than the traditional antidepressant
medications (Hashida et al. 2008). Numerous
studies have proven the clinical efficacy of
H. perforatum in both human and animal behav-
ioral models of depression (Hashida et al. 2008).
Hypericin (Fig. 8.2) is one of the main consti-
tuents of Hypericum species and was first isolated
from H. perforatum (Kusari et al. 2008). The com-
pound is responsible for photosensitivity of
patients during therapy with H. perforatum pre-
parations (Roth et al. 1994) and has only been
available in the plants of Hypericum species
(Kusari et al. 2008). Recently, an endophytic fun-
gus was isolated from the stems of H. perforatum,
which produced the naphthodianthrone derivate
hypericin and the putative precursor emodin
(Kusari et al. 2008). An unequivocally identifica-
tion of the fungal endophyte was not possible, but
molecular analysis revealed that it is related to
Chaetomium globosum (Kusari et al. 2008).
F. Podophyllotoxin
The lignan podophyllotoxin (Fig. 8.2) is highly
valued as a precursor to clinically useful anticancer
 Endophytic Fungi, Occurrence and Metabolites
drugs (Eyberger et al. 2006). The consequence of
the destruction of the primary source Podophyllum
emodi was an investigation of the endophytic fungi
of Podophyllum species (Eyberger et al. 2006; Puri
et al. 2006). Recently, Puri et al. (2006) and Eyber-
ger et al. (2006) described the production of podo-
phyllotoxin by Trametes hirsuta isolated from
P. emodi and Phialocephala fortinii from rhizomes
of the plant Podophyllum peltatum, respectively.
IV. Metabolites from
Endophytic Fungi
The review of Tan and Zou (2001) ‘‘Endophytes: a
rich source of functional metabolites’’ gives an
overview of endophytic metabolites characterized
before 2000. In the review ‘‘Natural products from
plant-associated microorganisms: distribution,
structural diversity, bioactivity, and implications
of their occurrence’’ of Gunatilaka (2006) 230
metabolites from over 70 plant-associated micro-
bial strains are discussed. Since these two articles
give a broad and comprehensive overview of the
metabolites from endophytic fungi, the com-
pounds from endophytic fungi in this article con-
centrate on publications of the past two years.
The following presents metabolites produced
by endophytic fungi from medicinal plants and
plants not used in medicine and describes the
medicinal plants and their traditional use in eth-
nopharmacology. Table 8.1 gives an overview of
the discussed endophytes, their host plants, and
their metabolites.
A. Metabolites from Endophytic
Xylariaceous Fungi
1. 7-Amino-4-Methylcoumarin from Xylaria
sp. of Ginkgo biloba
Traditional Chinese medicine uses the Ginkgo biloba
leaves for brain disorders, circulatory disorders, asthma,
and as an antiparasitic agent (Dugoua et al. 2006). G.
biloba is commonly used for the treatment of dementia,
but also for memory improvement, cerebrovascular and
aterial insufficiency, tinnitus, vertigo, asthma, and aller-
gies (Bent et al. 2005). The mode of action of this herb is
not completely clear, it is believed to improve cerebral and
peripheral blood flow through nitric oxid-induced vasodi-
latation (Bent et al. 2005). In addition, G. biloba possess
159
antioxidant, anti-inflammatory, and antitumor activities
(Bent et al. 2005; Chan et al. 2007).
Recently, Liu and coworkers (2008) reported the
isolation of an endophytic Xylaria sp. from
healthy twigs of G. biloba. Extracts of the fungal
culture fluid exhibited broad antimicrobial activity
and bioactivity-guided purification yielded
7-amino-4-methylcoumarin (Fig. 8.3; Liu et al.
2008). Strong antibacterial and antifungal activ-
ities of this coumarin derivate were observed
against several bacteria and fungi, respectively.
2. Metabolites from Xylaria sp. from
Sandoricum koetjape
Sandoricum koetjape Merr. is a medicinal plant widely
distributed in tropical areas of Asia (Ismail et al. 2003).
The bark and the whole plant are used in folk medicine as
a tonic after childbirth and for the treatment of colic and
leucorrhea in Malaysia and Indonesia (Ismail et al. 2003).
Phytochemical studies of this plant reported the isolation
of triterpenoids and limonoids from the heartwood, bark,
and seeds and the limonoids sandrapins A, B, and C from
the leaves (Ismail et al. 2004). Anti-inflammatory, ichthyo-
toxic, and anticarcinogenic effects were described for
extracts of S. koetjape and some of its constituents, respec-
tively (Ismail et al. 2004; Rasadah et al. 2004).
Tansuwan and coworkers (2007) isolated the
endophytic fungus PB-30 from healthy leaves of
S. koetjape collected from Prachinburi Province
in Thailand. The ITS sequence suggests that this
endophytic fungus belongs to the genus Xylaria
(Tansuwan et al. 2007). Two novel benzoquinone
metabolites, 2-chloro-5-methoxy-3-methylcyclo-
hexa-2,5-diene-1,4-dione and 7-hydroxy-8-meth-
oxy-3,6-dimethyldibenzofuran-1,4-dione, named
xylariaquinone A, together with the known natural
products 2-hydroxy-5-methoxy-3-methylcyclo-
hexa-2,5-diene-1,4-dione and 4-hydroxymellein
were obtained from extracts of fungal cultures
(Fig. 8.3; Tansuwan et al. 2007). The antimalarial
activity of the four compounds was tested against
Plasmodium falciparum. 2-Chloro-5-methoxy-
3-methylcyclohexa-2,5-diene-1,4-dione and xylaria-
quinone A exhibited IC50 values of 1.84 mM and 6.68
mM, whereas the two known compounds were
inactive (Tansuwan et al. 2007). The cytotoxic activ-
ity of the cyclohexa-diene-dione and xylariaquinone
A against African green monkey kidney fibroblasts
was investigated by using a colorimetric method and
the IC50 values were respectively determined as 1.35
mM and >184 mM (Tansuwan et al. 2007).
160 Daniela Weber

Table 8.1. Metabolites from endophytic fungi

Microbial strain Host plant; plant part or Metabolites

tissue
Alternaria sp. Polygonum senegalense; Alternariol 5-O-sulfate
fresh healthy leaves Alternariol 5-O-methyl ether-40 -O-sulfate
Alternariol
Alternariol 5-O-methylether
Altenusin
2,5-Dimetyl-7-hydroxy-chromone
Tenuazonic acid
Altertoxin I
30 -Hydroxyalternariol 5-O-methylether
Desmethylaltenusin
Alterlactone
Alternaric acid
Alternariol
Alternariol 5-O-methyl ether
Altenusin
Talaroflavone
40 -Epialtenuene
Alternaria sp. (P 0506) Vinca minor; leaf Altersetin
Alternariol
Alternariol monomethylether
Tenuazonic acid
Alternaria sp. (P 0535) Euonymus europaeus; fruit Altersetin
Alternariol
Alternariol monomethylether
Tenuazonic acid
Ascochyta sp. (6651) Meliotus dentatus (4S)-(+)-Ascochin
(S,S)-(+)-Ascodiketone
(3R,4R)-()-4-Hydroxymellein
Ent-a-cyperone
(3S,4R)-()-Dihydroxy-(6S)-undecyl-a-pyranone
Botryosphaeria mamane (PSU-M76) Garcinia mangostana; Botryomaman
leaves 2,4-Dimethoxy-6-pentylphenol
(R)-()-Mellein
Primin
Cis-4-hydroxymellein
Trans-4-hydroxymellein
4,5-Dihydroxy-2-hexenoic acid
Botryosphaeria parva Schinus molle; small woody 2,7-Dimethoxy-6-(1-acetoxyethyl)-juglon
twigs
Chaetomium sp. Nerium oleander L. Phenolics (e. g. phenolic acids and their derivates)
Volatile and aliphatic compounds
Chaetomium globosum (IFB-E019) Imperata cylindrica; stem Chaetoglobosin U
Chaetoglobosin C
Chaetoglobosin F
Chaetoglobosin E
Penochalasin A
Colletotrichum dematium Pteromischum sp. Colutellin
Colletotrichum gloeosporioides Artemisia mongolica Colletotric acid
Colletotrichum gloeosporioides Schinus molle; leaves DW E01141-1
(E01141)
Coniothyrium sp. (7303) Artemisia maritima Massarilactone E
Massarilactone F
Massarilactone G
Massarilactone acetonide
Diaporthe sp.
(continued)
 Endophytic Fungi, Occurrence and Metabolites 161

Table 8.1. (continued)

Microbial strain Host plant; plant part or Metabolites

tissue
Camellia sinensis L.; young (+)-Epicytoskyrin
stems (+)-1,10 -Bislunatin
Dothiodeomycete sp. (LRUB20) Leea rubra; stem 2-Hydroxymethyl-3-methylcyclopent-2-enone
Cis-2-hydroxymethyl-3-methyl-cyclo-pentanone
Asterric acid
Emericella variecolor Croton oblongifolius; Shamixanthone
mature petioles 14-Methoxytajixanthone-25-acetate
Taijixanthone methanoate
Tajixanthone hydrate
Endophytic ascomycete (related to Plant samples Phaeosphaeride A
Phaeosphaeria avenaria) Phaeosphaeride B
Epichloe typhina Phleum pretense L. Epichlicin
Fusarium sp. (CR377) Selaginella pallescens; stem CR377
Fusarium sp. (YG-45) Maackia chinensis; twig Fusaristatin A
segment Fusaristatin B
Hormonema dematioides Pinus sp.; needle base Hormonemate
Hypoxylon truncatum (IFB-18) Artemisia annua; stem Daldinone C
tissue Daldinone D
Altechromone A
(4S)-5,8-Dihydroxy-4-methoxy-a-tetralone
Leptosphaeria sp. (IV403) Artemisia annua Leptosphaeric acid
Mitosporic, hyaline fungus E-3 Prymnopitys andina; wood 4-(2-Hydroxyethyl)phenol
pieces p-Hydroxybenzaldehyde Mellein
Nodulisporium sp. Junipercus cedre; twigs 3-Hydroxy-1-(2,6-dihydroxyphenyl)-butan-1-one
1-(2-Hydroxy-6-methoxyphenyl)butan-1-one
2,3-Dihydro-5-methoxy-2-methylchomen-4-one
Nodulisporin A
Nodulisporin B
Nodulisporin C
(4E,6E)-2,4,6-Trimethyloacta-4,6-dien-3-one
1-(2,6-Dihydroxyphenyl)butan-1-one
5-Hydroxy-2-methyl-4H-chromen-4-one
2,3-Dihydro-5-hydroxy-2-methylchomen-4-one
8-Methoxynaphthalen-1-ol
1,8-Dimethoxynaphthalene
Daldinol
Helicascolide A
(1S,4S,4aS,6R,6aR)-Decahydro-1,4-dimethyl-6-
(prop-1-en-2-yl)naphthalene-1-ol
Ergosterol
5a,8a-Epidioxyergosterol
Penicillium sp. (IFB-E22) Quercus variabilis; stem Penicidone A
Penicidone B
Penicidone C
Lumichrome
Physicion
Emodin-1,6-dimethylether
Penicillium sp. (GQ-7) Aegiceras corniculatum L.; Penicillenol A1
inner bark Penicillenol A2
Penicillenol B1
Penicillenol B2
Penicillenol C1
Penicillenol C2
Citrinin
Phenol A acid
Phenol A
Dihydrocitrinin
(continued)
162 Daniela Weber

Table 8.1. (continued)

Microbial strain Host plant; plant part or Metabolites

tissue
Penicillium chrysogenum (No. 005) Cistanche deserticola; root Chrysogenamide A
Circumdatin G
2-[(2-Hydroxypropionyl) amino] benzamide
20 ,30 -Dihydrosorbicillin
(9Z,12Z)-2,3-Dihydroxypropyl octadeca-9,
12-dienoate
Penicillium janczewskii K.M.Zalesky Prymnopitys andina; wood Peniprequinolone
pieces Gliovictin
Penicillium janczewskii K.M.Zalesky Prymnopitys andina Pseurotin A
Cycloaspeptide A
Penicillium paxilli (PSU-A71) Garcinia atroviridis; leaves Penicillone
Paxilline
Pyrenocine A
Pyrenocine B
Phoma medicaginis Medicago sativa, Brefeldin A
M. lupulina
Phomopsis sp. Camptotheca acuminata 8-O-Acetylmultiplolide A
8-O-Acetyl-5,6-dihydro-5,6-epoxymultiplolide A
5,6-Dihydro-5,6-epoxymultiplolide A
3,4-Deoxy-3,4-didehydromultiplolide A
Multiplolide A
(4E)-6,7,9-Trihydroxydec-4-enoic acid
(4E)-6,7,9-Trihydroxydec-4-enoate
Phomopsis spp. Erythrina crista-galli; twigs, Clavatol
leaves Hydroxymellein
Mellein
Mevalonolactone
Mevinic acid
Nectriapyrone
Scytalone
Tyrosol
Phomol
DW E02018-5
DW E02018-9
Phomopyronol
3-Phenylpropane-1,2-diol
4-(2,3-Dihydroxypropoxy)benzoic acid
2-(Hydroxymethyl)-3-propylphenol
2-(Hydroxymethyl)-3-(1-hydroxy-propyl)-phenol
Compound 6
8-(Hydroxymethyl)-2,2-dimethyl-7-propyl-
chroman-3-ol
(4E,10E)-Trideca-4,10,12-triene-2,8-diol
Phomopsis sp. Eupatorium arnotianum; Phomopsidone
leaves, stems Mellein
Nectriapyrone
Phomopsis sp. Phytolacca dioica; twig Cytosporone D
Phomopsis sp. (BCC 1323) Tectonia grandis L.; leaf Phomaxanthone A
Phomaxanthone B
Phomopsis sp. (YM3111483) Azadirachate indica; fresh Multiplolide A
stems 8a-Acetoxy-5a-hydroxy-7-oxodecan-9-olide
7a,8a-Dihydroxy-3,5-decadien-10-olide
7a-Acetoxymultiplolide A
8a-Acetoxymultiplolide A
Phomopsis cassiae Cassia spectabilis; leaves Two diastereoisomeric
3,9,12-trihydroxycalamenenes
3,12-Dihydroxycalamenene
3,12-Dihydroxycadalene
3,11,12-Trihydroxycadalene
(continued)
 Endophytic Fungi, Occurrence and Metabolites 163

Table 8.1. (continued)

Microbial strain Host plant; plant part or Metabolites

tissue
Phomopsis longicolla Dicerandra frutescens; stem Dicerandrol A
segment Dicerandrol B
Dicerandrol C
Phyllosticta spinarum Platycladus orientalis; (+)-(5S,10S)-40 -Hydroxymethylcyclozonarone
healthy foliage 3-Ketotauranin
3a-Hydroxytauranin
12-Hydroxytauranin
Phyllospinarone
Tauranin
Pestalotiopsis sp. Pinus taeda Pestalotiopsolide A
Taedolidol
6-Epitaedolidol
Pestalotiopsis foedan Unidentified tree Pestafolide A
Pestaphthalide A
Pestaphthalide B
Pestalotiopsis microspora Terminalia morobensis; Isopectacin
small tree stem
Pestalotiopsis theae Unidentified tree Pestalotheol A
Pestalotheol B
Pestalotheol C
Pestalotheol D
Xylaria sp. Ginkgo biloba; healthy 7-Amino-4-methyl-coumarin
twigs
Xylaria sp. Sandoricum koetjape; 2-Chloro-5-methoxy-3-methylcyclohexa-2,
healthy leaves 5-diene-1,4-dione
Xylariaquinone A
2-Hydroxy-5-methoxy-3-methylcyclohexa-2,
5-diene-1,4-dione
4-Hydroxymellein
Xylaria sp. (no. 2508) Seed of angiosperm tree Xyloketal J
Xyloester A
Xyloallenolide B
Dihydrobenzofuran derivate
Xylariaceous endophyte (YUA-026) Twigs and petiols of plant Eremoxylarin A
samples Eremoxylarin B

3. Sesquiterpenoids from a Xylariaceous Fungus
Shiono and Murayama (2005) isolated the xylar-
iaceous endophyte YUA-026 from twigs and
petiols of plant samples collected from Mount
Takadate in Japan. Two new eremophilane-type
sesquiterpenoids, eremoxylarin A and B, were
obtained from rice culture of the strain YUA-026
(Fig. 8.3; Shiono and Muranyama 2005). The pu-
rification of the metabolites was guided by their
antimicrobial activity against Pseudomonas aeru-
ginosa and analytical methods (Shiono and Mur-
anyama 2005). Using agar dilution methods, the
minimum inhibition concentration (MIC) of ere-
moxylarins A and B were evaluated against the
gram-positive bacterium Staphylococcus aureus
(12.5, 25.0 mg/ml), the gram-negative bacterium
Pseudomonas aeruginosa (6.25, 12.5 mg/ml), and
the fungi Candida albicans and Aspergillus clava-
tus (Shiono and Muranyama 2005). The MICs of
the two eremoxylarins against the two tested
fungi were over 100 mg/ml (Shiono and Mura-
nyama 2005).
4. Metabolites from Xylaria sp.
The ascomycete Xylaria sp. (2508) was isolated
from seed of an angiosperm tree in Mai Po man-
grove, Hong Kong (Xu et al. 2008). Fermentations
of fungus yielded three new metabolites, named
xyloketal J, xyloester A, and xyloallenolide B,
164 Daniela Weber

O
O
O
O

H OH
Cl H
H2N O O O
O H O
2-Chloro-5-methoxy-3-
7-Amino-4-methylcoumarin methylcyclohexa-2,5- Xylariaquinone A
diene-1,4-dione

O
O OH O
O

O
HO H
O CH3
2-Hydroxy-5-methoxy- OH
3-methylcyclohexa-2,5 O
4-Hydroxymellein HO O
-diene-1,4-dione

O H H
O

O
O
HO
O
O Xyloketal J

Xyloester A

O H O OH Eremoxylarin A R=

H O

H
O Eremoxylarin B R=
H OR
O

Fig. 8.3. Metabolites from endophytic xylariaceous fungi

along with the known substituted dihydrobenzo-
furan (Figs. 8.3, 8.4; Xu et al. 2008). The structures
of all the compounds possess a substituted dihy-
drobenzofuran unit (Xu et al. 2008).
B. Metabolites from Endophytic Phomopsis
or Diaporthe Species
1. Lactones from Phomopsis sp. from
Azadirachata indica
Neem (Azadirachata indica) is used as a traditional me-
dicinal plant in India (Biswas et al. 2002). Each part of the
tree is used as traditional medicine, e.g. the leaves, twigs,
flowers, and fruits are used for the elimination of intestinal
worms, and the bark is used as an analgesic and curative of
fever (Biwas et al. 2002). Neem preparations are used eco-
nomically as insecticides, pesticides, and agrochemicals
(Brahmachari 2004). Neem seed extracts provide a poten-
tial control of Anopheles larvae that could be complemen-
tary to other eradication methods (Gianotti et al. 2008).
Wu et al. (2008) isolated the endophytic fungus
Phomopsis sp. YM311483 from fresh stems of an
apparently healthy Azadirachata indica collected
in Yunnana Province, China. Four new 10-
membered lactones and the known lactone,
multiplolide A (Fig. 8.4), were isolated from the
culture broth of this Phomopsis strain. The new
 Endophytic Fungi, Occurrence and Metabolites 165

CH2OH
O
C
O O O

Dihydrobenzofuran derivate Xyloallenolide B

OR1 OH O OH
OR2 OAc OH
O
O O O

O O O
Multiplolide A R1=R2=H
8a-Acetoxymultiplolide A R1=H, R2=Ac 8a-Acetoxy-5a-hydroxy- 7a,8a-dihydroxy-3,5-
7-oxodecan-9-olide decadien-10-olide
7a-Acetoxymultiplolide A R1=Ac, R2=H

OH O OH

OH
HO
O
O
H3CO OH HO O
HO OCH3
O
O
O
O HO O
OH
HO

(+)-Epicytoskyrin
OH O OH

(+)-1,1´-Bislunatin

Fig. 8.4. Metabolites from endophytic xylariaceous fungi and from endophytic Phomopsis and Diaporthe species

compounds were determined as 8a-acetoxy-5a-
hydroxy-7-oxodecan-9-olide, 7a,8a-dihydroxy-
3,5-decadien-10-olide, 7a-acetoxymultiplolide A,
and 8a-acetoxymultiplolide A (Wu et al. 2008).
Antifungal activity against seven plant pathogens
was examined using the paper disk diffusion
method. All five metabolites showed weak anti-
fungal activity, whereas 8a-acetoxymultiplolide A
was the most potent, with MIC values of 31.25–
500.0 mg/ml (Wu et al. 2008).
2. Metabolites from Diaporthe sp. from
Camellia sinensis L.
Green, black, and oolong teas are prepared from leaves
of Camellia sinensis L., a member of the family Theaceae
cultivated widely in China, India, Japan, and Indonesia
(Brown 1999, Luper 1999). It is the production process
which makes the difference of the types of tea (Brown
1999). Consumption of tea (C. sinensis) is suggested to
prevent cancer, heart disease, and other diseases
(Lambert and Yang 2003). Yang et al. (2001) describe
tea as one of the few chemoprotective agents known to
have protective effects at different stages of the carcino-
genic process. It is assumed that tea constituents (like
polyphenolic components) may inhibit this process by
modulating signal transduction pathways leading to the
inhibition of cell proliferation and transformation and
enhancement of apoptosis (Yang et al. 2001). In addition,
hepatoprotective activity against a variety of toxic com-
pounds (e.g. alcohol) and detoxification activity by en-
hancement of glucuronidation in the liver are described
for tea (Luper 1999). The antimicrobial activity against
a variety of bacterial species (Staphylococcus, Salmonella,
Shigella, Vibrio, and others; Hamilton-Miller 1995) may
166 Daniela Weber
explain the observed reduction of incidence and severity
of caries by regular tea drinking in a limited number of
clinical trails in man (Hamilton-Miller 2001).
Agusta and coworkers (2005) isolted 35 filamen-
tous endophytic fungi from young stems of C.
sinensis collected in West Java, Indonesia. These
fungi were classified into six species by morpho-
logical considerations and RAPD analysis
(Agusta et al. 2005). One isolate, a Diaporthe
sp., was able to transform catechins with a 2R-
phenyl substitution into the corresponding 3,4-
cis-dihydroxyflavan derivatives (Agusta et al.
2006). A second Diaporthe sp., closely related to
Diaporthe phaseolorum srain sw-93-13, produced
two bisanthraquinones named (+)-epicytoskyrin
and (+)-1,10 -bislunatin (Fig. 8.4, Agusta et al.
2006). (+)-Epicytoskyrin is an epimer of cytos-
kyrin A, which was isolated from an endophytic
Cytospora sp. isolated from Conocarpus erecta
(Brady et al. 2000b). (+)-1,10 -Bislunatin is a
homodimeric compound of lunatin joined to-
gether at C-1 and C-10 and the relative structure
of bislunatin has been reported from Verticillium
lecanii (Agusta et al. 2006). (+)-Epicytoskyrin
and (+)-1,10 -bislunatin showed a moderate cyto-
toxic activity against KB cells, with an IC50 value
of 0.5 mg/ml and 3.5 mg/ml, respectively (Agusta
et al. 2006).
3. Metabolites from Phomopsis sp. from
Camptotheca acuminata
The antitumor agent camptothecin was originally
isolated from the medicinal plant Camptotheca
acuminata. Lin et al. (2007) isolated 174 endo-
phytic fungi from this medicinal plant. From fer-
mentations of one strain, which was identified by
morphologic and genetic properties as Phomopsis
sp., several new compounds were purified, includ-
ing the 10-membered macrolides 8-O-acetylmulti-
plolide A, 8-O-acetyl-5,6-dihydro-5,6-epoxymulti-
plolide A, 5,6-dihydro-5,6-epoxymultiplolide A,
and 3,4-deoxy-3,4-didehydromultiplolide A, and
their known parent compound multiplolide A
(Figs. 8.4, 8.5), as well as the ansaturated fatty
acid (4E)-6,7,9-trihydroxydec-4-enoic acid and its
methyl ester (Fig. 8.5; Tan et al. 2007). The 10-
membered macrolides and multiplolide A exhihib-
ited no evident antifungal activities against Candi-
da albicans.
4. Sesquiterpenoids from Phomopsis cassiae
from Cassia spectabilis
Several species of Cassia are widely used in tradi-
tional medicine for their antimicrobial, laxative,
antiulcerogenic, analgesic, and anti-inflammatory
properties (Viegas et al. 2008). The endophytic
fungus Phomopsis cassiae was isolated from leaves
of the Leguminosae Cassia spectabilis collected in
Brazil (Silva et al. 2006). Bioassay-guided fraction-
ation of crude extracts of the fungal strain resulted
in the isolation of the two diastereoisomeric
3,9,12-trihydroxycalamenenes, 3,12-dihydroxyca-
lamenene, 3,12-dihydroxycadalene, and 3,11,12-
trihydroxycadalene (Fig. 8.5; Silva et al. 2006). Of
these new cadinane sesquiterpenoids, 3,11,12-tri-
hydroxycadalene, was the most active against the
phytopathogenic fungi Cladosporium sphaeros-
permum and C. cladosporioides (Silva et al.
2006). 3,12-Dihydroxycalamenene, 3,12-dihy-
droxycadalene, and 3,11,12-trihydroxycadalene
exhibited cytotoxic activity against the human
cervical tumor cell line HeLa with IC50 values
of 100, 20, and 110 mmol/l, respectively (Silva
et al. 2006).
5. Metabolites from Phomopsis spp. from
Erythrina crista-galli
Erythrina crista-galli (Fabaceae) is used in Argen-
tinean ethnopharmacology as anti-inflammatory
medication, narcotic, desinfectant, and for the
treatment of wounds. Anti-inflammatory (Miño
et al. 2002) and antibacterial (Mitscher et al.
1988) activities have been described for E. crista-
galli. This Argentinean medicinal plant can be
found in the tropical and subtropical regions of
America and is commonly used as an ornamental
plant. In Argentina the wood is used in infusions
or decoctions as astringent, narcotic, and sedative
(Toursarkissian 1980).
The majority of the fungi isolated so far from
different collections of E. crista-galli belong to the
genus Phomopsis (Weber et al. 2005). This genus
comprises a multitude of species widely
distributed as pathogens, endophytes or even
symbionts of plants (Uecker 1988). The metabo-
lites of 12 different Phomopsis isolates were
investigated and from four strains metabolites
were isolated. Besides the known compounds
clavatol, hydroxymellein, mellein, mevalonolac-
tone, mevinic acid, nectriapyrone, scytalone, and
 Endophytic Fungi, Occurrence and Metabolites 167

OH OH OH
O
OAc OR OH

O O
O O O

O O O

8-O-Acetylmultiplolide A 8-O-Acetyl-5,6-dihydro-5,6- R=Ac 3,4-Deoxy-3,4-didehydro-

epoxymultiplolide A R=H multiplolideA
5,6-Dihydro-5,6-epoxymulti-
plolide A

OH
HO OH
OR

OH OH O
HO

(4E)-6,7,9-trihydroxydec-4-enoic acid R=H (-)-(7S,9S,10S)-3,9,12-

(4E)-6,7,9-trihydroxydec-4-enoate R=Me Trihydroxycalamenene

HO OH HO HO

HO HO HO

(-)-(7S,9R,10S)-3,9,12- 3,12-Dihydroxy 2,12-Dihydroxycadalene

Trihydroxycalamenene calamenene

O OH
HO
OH
O O

HO OH O OH O
OH OH

3,11,12-Trihydroxycadalene Clavatol 4-Hydroxymellein Mellein

Fig. 8.5. Metabolites from endophytic Phomopsis species from Camptotheca acuminata, Cassia spectabilis, and
Erythrina crista-galli

tyrosol (Figs. 8.5, 8.6), the new compounds pho-
mol, two derivates of phomol, DW E02018-5 and
DW E02018-9, phomopyronol, 3-phenylpropane-
1,2-diol, 4-(2,3-dihydroxypropoxy)benzoic acid,
2-(hydroxymethyl)-3-propylphenol, 2-(hydroxy-
methyl)-3-(1-hydroxypropyl)phenol, compound
6, 8-(hydroxymethyl)-2,2-dimethyl-7-propylchro-
man-3-ol, and (4E,10E)-trideca-4,10,12-triene-
2,8-diol were identified (Figs. 8.6, 8.7; Weber
et al. 2004, 2005; Weber 2006).
Mellein was described from fermentations of
Aspergillus melleus (Nishikawa 1933), Fusarium
larvarum (Grove and Pople 1979), Cercospora tai-
wanensis (Camarda et al. 1976), and many other
fungi. The compound has phytotoxic, antibacter-
ial, and antifungal activities (Takeuchi et al. 1992;
Wenke 1993). Mellein and nectriapyrone were
isolated by Claydon et al. (1985) from Phomopsis
oblonga, commonly found on the bark of trees of
the genus Ulmus. Trees infected by P. oblonga are
168 Daniela Weber

HO O

O
O O
O

O
O
OH
O O

Mevalonolactone Mevinic acid Nectriapyrone

OH OH
OH O
HO O

O
HO O
HO OH O

OH

Scytalone Tyrosol Phomol

OH
OH HO O
HO OH
O
O
O
O
O

DW E02018-5 DW E02018-9

OH
OH
O O OH

Phomopyronol 3-Phenylpropane-1,2-diol

Fig. 8.6. Metabolites from endophytic Phomopsis species from Erythrina crista-galli

protected from the attack of insects of the genus
Scolytus (bark beetle; Webber 1981).
Moderate or no antimicrobial, nematicidal, or
cytotoxic activities were observed for the new
compounds isolated from Phomopsis spp.
(Weber et al. 2005; Weber 2006). In the mouse
ear assay however, two compounds, mevinic
acid and phomol, showed significant activities
(Weber et al. 2004, 2005). Mevalonic acid is an
intermediate in cholesterol biosynthesis. Mevinic
acid is an inhibitor of HMG-CoA reductase, the
key enzyme of cholesterol biosynthesis (Endo and
Hasumi 1997). Derivatives are used as cholesterol-
lowering drugs. Phomol was the only fungal com-
pound, which was detected in small amounts in
plant extracts of E. crista-galli by HPLC-MS
analysis and thus can contribute to the anti-
inflammatory activity of the plant (Weber 2006).
 Endophytic Fungi, Occurrence and Metabolites 169

OH
OH OH OH
O OH HO
HO
O

HO

4-(2,3-Dihydroxypropoxy)- 2-(Hydroxymethyl)-3- 2-(Hydroxymethyl)-3-

benzoic acid propylphenol (1-hydroxypropyl)phenol

OH OH

O
O
OH (4E,10E)-Trideca-4,10,12-triene-2,8-diol

Compound 6

OH
O
O
O

HO
HO O
O
HO 8-(Hydroxymethyl)-2,2-dimethyl-
HO 7-propylchroman-3-ol
O

Phomopsidone

OH O OH

O OH

O O
OH
OAc OAc AcO OAc
OH
O

OH O OH

Cytosporone D Phomaxanthone A

Fig. 8.7. Metabolites from endophytic Phomopsis species from Erythrina crista-galli, Eupatorium arnottianum, Phyto-
lacca dioica, and Tectona grandis L.

6. Metabolites from Phomopsis sp. from Clavin et. al. 2000b), and antimicrobial activities (Penna et.
Eupatorium arnottianum al. 1997).

An endophytic Phomopsis was isolated from plant

Eupatorium arnottianum occurs in the northeast and cen-
ter of Argentina and in the south of Bolivia and is used for
the treatment of gastric pains (Iharlegui and Hurell 1992),
asthma, bronchitis, and colds (Girault 1987; Clavin et. al.
1999). Infusions of E. arnottianum showed analgesic (Cla-
vin et. al. 2000a), antiviral (against Herpes simplex type 1;
material collected in Argentina (Meister et al. 2007).
Besides mellein and nectriapyrone (Figs. 8.5, 8.6),
cultures yielded phomopsidone, a novel depsidone
derivate (Fig. 8.7; Meister et al., 2007). The
new metabolite showed no antimicrobial, cytotoxic,
170 Daniela Weber
nematicidal, or phytotoxic activities (Meister
et al. 2007).
7. Cytosporone D from Phomopsis sp.
of Phytolacca dioica
Phytolacca dioica is used in ethnopharmacology as
a vulnerary, vermifuge, and antipyretic medicine (Quiroga
et al. 2001). Aqueous and alcoholic extracts from P. dioica
showed antimicrobial activity against gram-positive and
gram-negative microorganisms (Quiroga et al. 2001). Phy-
tolacca species are used as antirheumatics and have mol-
luscicidal activity (Soliman et al. 2001).
The known metabolite cytosporone D was isolated
from an endophytic Phomopsis sp., isolated from a
twig of P. dioica (Fig. 8.7; Weber 2006). The anti-
bacterial octaketide was already described from
Cytospora sp. and Diaporthe sp., which were
isolated as endophytes from Conocarpus erecta
and Forsteronia spicata (Brady et al. 2000a).
8. Xanthone Dimers from Phomopsis sp. from
Tectona grandis L.
The endophyte Phomopsis sp. BCC 1323 was
isolated from a teak leaf of Tectona grandis
L. collected in Northern Thailand. (Isaka et al.
2001). The search for bioactive and diverse meta-
bolites resulted in the isolation and identification
of two new xanthone dimers, phomaxanthones A
and B (Figs. 8.7, 8.8; Isaka et al. 2001). The two
compounds exhibited activity against Plasmodi-
um falciparum (K1, multidrug-resistant strain)
and against Mycobacterium tuberculosis (H37Ra
strain) with IC50 values of 0.11–0.33 mg/ml and
0.50–6.25 mg/ml, respectively (Isaka et al. 2001).
The deacetylated derivate of phomaxynthone A
was inactive in all assays.
9. Dicerandrols from Phomopsis longicolla
from the Mint Dicerandra frutescens
Dicerandra frutescens is a rare mint plant that
showed to be free of injury from insects (Wagen-
aar and Clardy 2001). A fungus identified as Pho-
mopsis longicolla by ITS sequencing was isolated
from a stem segment of D. frutescens (Wagenaar
and Clardy 2001). Bioassay-guided fractionation
yielded three related antibiotics, named diceran-
drols A, B, and C (Fig. 8.8). These new nat-
ural products are structurally related to the
ergochromes and secalonic acids and exhibited
antibacterial activity against Staphylococcus aure-
us and Bacillus subtilis as well as moderate activity
against human cancer cell lines (A549 and HCT-
116; Wagenaar and Clardy 2001).
C. Metabolites from Endophytic
Penicillium Species
1. Metabolites from Penicillium sp. from
Aegiceras corniculatum L.
Aegiceras corniculatum L. is used as a traditional medicine
for treatment of inflammation and liver injury (Roome
et al. 2008). Furthermore Dahdouh-Guebas and coworkers
(2006) reported that the bark of A. corniculatum is con-
verted into a paste and used as a fish poison.
Lin et al. (2008a) isolated the endophytic Penicil-
lium sp. GQ-7 from the inner bark of A. cornicu-
latum collected in China. They were able to purify
six new tetramic acids derivatives, along with the
mycotoxin citrinin, phenol A acid, phenol A, and
dihydrocitrinin from ethyl acetate extracts of Pen-
icillium sp. (Fig. 8.9). The six new compounds
were named penicillenols A1, A2, B1, B2, C1, and
C2 (Fig. 8.8; Lin et al. 2008a). The cytotoxic activity
of the penicillenols was tested against A-549, BEL-
7402, P388, and HL-60 cells by the MTT method.
Penicillenols C1 and C2 showed no cytotoxicity,
whereas the other four compounds exhibited
cytotoxic activity with IC50 values ranging from
0.76 mM to 16.26 mM against HL-60 cells (Lin et al.
2008a).
2. Metabolites from Penicillium chrysogenum
from Cistanche deserticola
Species of the genus Cistanche are used in tradi-
tional medicine and are also known as ‘‘Ginseng
of the desert’’. The Cistanche herbs are used as a
superior tonic for the treatment of kidney defi-
ciency, impotence, female infertility, morbid leu-
corrhea, profuse metrorrhagia, and senile
constipation (Jiang and Tu 2009). Cistanche deser-
ticola is a holoparasitic plant and a valuable tradi-
tional Chinese medicine (Dong et al. 2007).
Penicillium chrysogenum No. 005 was isolated
from the root of Cistanche deserticola collected
from Inner Mangolia in northwest China (Lin
et al. 2008b). A new member of the marcfortine
 Endophytic Fungi, Occurrence and Metabolites 171

O
R1
O O
O
OH O OH

OH O OH
HO O
OH O OH
O O
O
R2
O
HO O
OAc O Dicerandrol A R1=R2=H
AcO OAc Dicerandrol B R1=Ac, R2=H
OAc Dicerandrol C R1=R2=Ac

Phomaxanthone B

O OH O OH

N N

O O
OH Penicillenol A1 OH Penicillenol A2

O
O
OH
OH
N
N

H
O
O
H Penicillenol B2
Penicillenol B1

O OH O OH

N N

O O
OH OH
Penicillenol C1 Penicillenol C2

Fig. 8.8. Metabolites from endophytic Phomopsis species from Tectona grandis L. and Dicerandra frutescens and from
Penicillium sp. from Aegiceras corniculatum L.

alkaloids, named chrysogenamide A (Fig. 8.9), was
isolated from the culture broth of P. chrysogennum
005 (Lin et al. 2008b). In addition, four known
compounds were isolated and identified as cir-
cumdatin G, 2-[(2-hydroxypropionyl)amino]ben-
zamide, 20 ,30 -dihydrosorbicillin, and (9Z,12Z)-2,3-
dihydroxypropyl octadeca-9,12-dienoate by com-
parision of their spectroscopic data with literature
data (Fig. 8.9; Lin et al. 2008b). Chrysogenamide A
showed a neurocyte-protecting effect against oxi-
dative stress-induced cell death in SH-SY5Y cells
(Lin et al. 2008b).
3. Metabolites from Penicillium spp. from
Prumnopitys andina
Schmeda-Hirschmann and coworkers (2005)
isolated two endophytic fungi from wood pieces
172 Daniela Weber

OH OH
OH
O O
O O
O
OH OH

Citrinin Dihydrocitrinin

HN O
N
NH
O N
O

N NH

OH

Chrysogenamide A
Circumdatin G

O OH O

NH2

NH HO
OH
O

2´,3´-Dihydrosorbicillin
2-[(2-hydroxypropionyl)-
amino]benzamide

OH

OH

(9Z,12Z)-2,3-Dihydroxypropyloctadeca-9,12-dienoate

Fig. 8.9. Metabolites from endophytic Penicillium species from Aegiceras corniculatum L. and Cistanche deserticola

of Prumnopitys andina (Lleuque). The mitosporic
and hyaline fungus E-3, which could not be identi-
fied, yielded 4-(2-hydroxyethyl)phenol, p-hydroxy-
benzaldehyde, and the isochromanone mellein
(Fig. 8.10; Schmeda-Hirschmann et al. 2005). Peni-
prequinolone and gliovictin were obtained from
the second fungus (Fig. 8.10), which was identified
as Penicillium janczewskii K.M. Zalessky. The
metabolites from the fungus E-3 exhibited weak
activity against several gram-negative and -positive
bacteria (Schmeda-Hirschmann et al. 2005). The
known compounds, peniprequinolone and
gliovictin, showed cytotoxic activity against AGS
cells (IC50 = 89 mM, 475 mM) and fibroblasts
 Endophytic Fungi, Occurrence and Metabolites 173

OH
O H

OH O

OH
OH

4-(2-Hydroxyethyl)- p-Hydroxybenz-
Mellein
phenol aldehyde

OH O
O
O NH
OH
OH OH
O
O
O OH

N O

H
Pseurotin A
Peniprequinolone

O HN N
O
S
O
N
OH NH
N
HN
S
O N
O O

Gliovictin

OH

Cycloaspeptide A

Fig. 8.10. Metabolites from endophytic Penicillium species from Prumnopitys andina

(IC50 = 116 mM, 681 mM) and antibacterial activity
against Bacillus brevis and B. subtilis in the agar
diffusion assay (Schmeda-Hirschmann et al. 2005).
Recently, Schmeda-Hirschmann and coworkers
(2008) reported the purification of pseurotin A
and cycloaspeptide A (Fig. 8.10) from cultures of
P. janczewskii K.M. Zalessky isolated from
P. andina. The two known compounds have been
described for several other Penicillium species, like
the psychrotolerant species P. jamesonlandense or
P. soppii (Frisvad et al. 2006). For pseurotin
A, which is known as a mono amine oxidase
inhibitor, effective nematicidal activity against
Bursaphelenchus xylophilus was described, but it
174 Daniela Weber

did not show any nematicidal activity against Pra-
tylenchus penetrans and Caenorhabditis elegans
(Hayashi et al. 2007). Cycloaspeptide A exhibited
moderate activity (IC50 = 3.5 mg/ml) against Plas-
modium falciparum (Dalsgaard et al. 2005). Pseur-
otin A exhibited moderate antimicrobial activity
and moderate cytotoxic activity against human
lung fibroblasts with an IC50 value of 1000 mM,
whereas the IC50 value of cycloaspeptide A was
>1000 mM (Schmeda-Hirschmann et al. 2008).
4. Penicidones from Penicillium sp. from
Quercus variabilis
The three new and cytotoxic g-pyridone alkaloids,
penicidones A-C (Fig. 8.11), were isolated from
extracts of the endophytic fungus Penicillium sp.
IFB-E22 from the stem of Quercus variabilis
(Ge et al. 2008). The three new compounds,
along with the known metabolites lumichrome,
physicion, and emodin-1,6-dimethylether, were
purified from the extracts (Fig. 8.11). One of
the compounds was identified as (R,E)-5-(5,7-
dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-
2-(prop-1-enyl)pyridin-4(1H)-one, shortly named
penicidone A, and the two others penicidones B
and C (Ge et al. 2008). The penicidones exhibited
moderate cytotoxicity against SW1116, K562, KB,
and HeLa cells with IC50 values between 21.11 mM
and 80.8 mM. The biosynthesis of the penicidones
is thought via a polyketide pathway together with
several biotransformations like cyclization and
transamination (Ge et al. 2008).
D. Metabolites from Endophytic
Alternaria Species
1. Metabolites from Alternaria sp. from
Polygonum senegalense
Polygonum species are known in traditional medicine for
their diuretic, cholagic, antihemorrhagic, and antiseptic
properties (Aly et al. 2008). P. senegalense is a traditional

O O O
O O O O

N O O N
RO O
H H

Penicidone A R=Me Penicidone C

Penicidone B R=H

O
H
Me N N O O Me

NH
Me N

O OH O OH

Lumichrome Physcion

OH O O

O Me

Fig. 8.11. Metabolites from en-

dophytic Penicillium sp. from
Quercus variabilis Emodin-1,6-dimethylether
 Endophytic Fungi, Occurrence and Metabolites
medicinal plant growing in Egypt which is used for the
treatment of skin diseases.
The endophyte Alternaria sp. was isolated from
fresh healthy leaves of P. senegalense collected
near Alexandria, Egypt (Aly et al. 2008). In liquid
Wickerham medium this fungus produced new
sulfated derivatives of alternariol and its mono-
methyl ethers (alternariol 5-O-sulfate and alter-
nariol 5-O-methyl ether-40 -O-sulfate) besides the
known compounds alternariol, alternariol 5-O-
methylether, altenusin, 2,5-dimetyl-7-hydroxy-
chromone, tenuazonic acid, and altertoxin I
(Fig. 8.12; Aly et al. 2008). From solid rice cultures
the four new compounds 30 -hydroxyalternariol 5-
O-methylether, desmethylaltenusin, alterlactone,
and alternaric acid together with alternariol, alter-
nariol 5-O-methyl ether, altenusin, talaroflavone,
and a new stereoisomer of altuene, named 40 -
epialtenuene, were obtained (Aly et al. 2008).
The cytotoxic activity of the isolated compounds
was tested against L5178Y mouse lymphoma cells.
Alternariol was the most active compound and
exhibited an EC50 value of 1.7 mg/ml, whereas
alternariol 5-O-sulfate and alternariol 5-O-methyl
ether showed EC50 values of 4.5 mg/ml and 7.8 mg/
ml, respectively (Aly et al. 2008). Alternariol 5-O-
methyl ether, altenusin, and alternariol were
detected in crude MeOH extracts of the host
plant P. senegalense by LC-MS (Aly et al. 2008).
2. Metabolites from Alternaria spp. from
Vinca minor and Euonymus europaeus
The endophytic Alternaria species P 0506 and P
0535 were isolated from a leaf of Vinca minor and
from a fruit of Euonymus europaeus, respectively
(Hellwig et al. 2002).
The species of the genus Vinca are well known because of the
isolation of the vinca alkaloids, e.g. vincristine or vinblastine.
These alkaloids bind specifically to tubulin, and therefore the
polymerization of the microtubli and the formation of the
spindle apparatus are blocked, resulting in an inhibition of
mitosis (Aktories et al. 2005). The vinca alkaloids are used for
the treatment of various tumors like malign lymphomas or
acute leukemia.
Several species of the genus Euonymus are used as
traditional medicinal plants, e.g. E. hederaceus or E. alatus
(Hu et al. 2005; Park et al. 2005). E. alatus has been used
for the treatment of diabetes and tumors in China and
Korea (Kim et al. 2006; Fang et al. 2008). E. europaeus is
the sole representative of this genus in central and western
Europe (Descoins et al. 2002). The fruits of the plant are
175
toxic for insects and ingestion of the fruits induces gastro-
intestinal discomfort in humans (Roth et al. 1994).
The two Alternaria species from V. minor and
E. europaeus produced similar secondary metabo-
lites in shake flasks and stirred fermentors (Hellwig
et al. 2002). A new metabolite, named altersetin was
produced together with the known metabolites alter-
nariol, alternariol monomethylether, and tenuazonic
acid by both fungal strains (Fig. 8.12; Hellwig et al.
2002). Altersetin exhibited antibacterial activity
against various human pathogenic bacteria in the
serial agar dilution assay (Hellwig et al. 2002).
E. Xanthones from Emericella variecolor from
Croton oblongifolius
Emericella variecolor was isolated from mature
petioles of the Thai medicinal plant Croton oblon-
gifolius (Pornpakapul et al. 2006). Four xanthones
were isolated from the mycelium of the endophytic
fungus. The metabolites were identified by spectro-
scopic analysis as shamixanthone, 14-methoxyta-
jixanthone-25-acetate, taijixanthone methanoate,
and tajixanthone hydrate (Fig. 8.13). The cytotoxic
activitiy of the four compounds was tested
against several human tumor cell lines and only
tajixanthone hydrate exhibited modest activity
against all tested cell lines including gastric carci-
noma (KATO3; IC50 = 10.9 mM), colon carcinoma
(SW620; IC50 = 13.6 mM), breast carcinoma (BT474;
IC50 = 12.3 mM), human hepatocarcinoma (HEP-
G2; IC50 = 16.4 mM), and lung carcinoma (CHAGO;
IC50 = 11.6 mM; Pornpakapul et al. 2006).
F. Cyclopentenons from Dothideomycete
sp. from Leea rubra
The endophyte Dothideomycete sp. LRUB20 was
isolated from the stem of the Thai medicinal plant
Leea rubra (Chomcheon et al. 2006). Two new
natural products, 2-hydroxymethyl-3-methylcy-
clopent-2-enone (synthetically known) and cis-
2-hydroxymethyl-3-methylcyclopentanone, and
the known compound asterric acid were isolated
from extracts of the culture broth of the endo-
phyte (Fig. 8.13; Chomcheon et al. 2006). In the
microplate Alamar Blue assay 2-hydroxymethyl-
3-methylcyclopent-2-enone and asterric acid
exhibited weak antimicrobial activity against
Mycobacterium tuberculosis with a MIC value of
200 mg/ml (Chomeon et al. 2006).
176 Daniela Weber

O
O
R1 O OH
HO HO OH

R 2O
HO

OR3 OR

R1=H, R2=H, R3=SO3H Altenusin R=CH3

Alternariol 5-O-sulfate
R =H, R =SO H, R =CH Desmethylaltenusin R=H
Alternariol 5-O-methylether-4´-O-sulfate 1 2 3 3 3
Alternariol R1=H, R2=H, R3=H
R1=H, R2=H, R3=CH3
Alternariol 5-O-methylether R1=OH, R2=H, R3=CH3
3´-Hydroxyalternariol 5-O-methylehter

OH O

O
HO O HO
H OH
OH
O
N
H H
O

OH O

2,5-Dimethyl-7-hydroxychromone Tenuazonic acid Altertoxin I

O OH
OH O
O OH
O
OH O
O
O OH
HO
O O
O
OH
HO
Alterlactone Alternaric acid Talaroflavone

O OH
HN
OH

R1 O OH
HO
R2
H
HO O

H
4´-Epialtenuene R1=H, R2=OH
Altenuene R1=OH, R2=H Altersetin

Fig. 8.12. Metabolites from endophytic Alternaria species

G. Metabolites from Endophytic therapeutically and as an instrument of suicide since the

Chaetomium Species antiquity (Langford and Boor 1996). The pink oleander,
Nerium oleander, is one of the principle oleander repre-
sentatives (Langford and Boor 1996). Certain parts of the
1. Metabolites from Chaetomium plant are used in Chinese folk medicine (Huang et al.
sp. from Nerium oleander L. 2007). N. oleander is supposed to have cardiotonic, anti-
bacterial, antileprotic, anti-inflammatory, anticancer, and
antiplatelet aggregation activities, insecticidal activity,
Oleanders (Apocynaceae), which contain the inotropic mammalian cytotoxicity, and act as a depressant of the
cardenolides (e.g. oleanderin), have been exploited central nerve system (Huang et al. 2007).
 Endophytic Fungi, Occurrence and Metabolites 177

H O
O
O
O

O
O
O OH
H OH O OH
H H OAc
H H
H
H
H

Shamixanthone 14-Methoxytajixanthone-25-acetate

OH
H
OH
H H
OH
H
O O
O

O
O O OH
H OH
O OH H
H OH
H H
H
H
H

Tajixanthone methanoate Tajixanthone hydrate

O
OH
HO HO

HO O
O O

HO O O O

2-Hydroxymethyl-3-methyl- cis-2-Hydroxymethyl-3- Asterric acid

cyclopent-2-enone methylcyclopentenone

Fig. 8.13. Metabolites from endophytic Emericella variecolor and Dothideomycete sp.

Huang and coworkers (2007) isolated, among 42
endophytic fungi, a Chaetomium sp. with the
strong antioxidant capacity from N. oleander.
The main bioactive metabolites from the fungal
cultures were phenolics (e.g. phenolic acids and
their derivates) and volatile and aliphatic com-
pounds which were detected by LC-ESI-MS and
GC-MS (Huang et al. 2007). Huang et al. (2007)
were not able to find the same compounds in the
host plant and its endophytic fungi.
2. Cytochalasan Alkaloids from Chaetomium
globosum from Imperata cylindrica
The endophyte Chaetomium globosum IFB-E019
was isolated from the stem of Imperata cylindrica
178 Daniela Weber

Me
O human nasopharyngeal epidermoid tumor KB cell
line. Chaetoglobosin U was the most cytotoxic,
Me
Me
with an IC50 value of 16 mM, whereas the four
H H
known compounds exhibited moderate activity
Me with IC50 values of 34–52 mM (Ding et al. 2006).
HN
O H

N
O
O OH
H. Metabolites from Endophytic
H Pestalotiopsis Species
Chaetoglobosin U
1. Isopestacin from Pestalotiopsis
O
microspora from Terminalia morobensis
Me

Me Species of the genus Terminalia are used as medicinal

Me
H
plants, e.g. the bark of Terminalia arjuna has a long history
of use as a cardiac tonic and is indicated for the treatment
Me
HN
of coronary artery disease, heart failure, hypercholesterol-
O emia, and for relief of anginal pain (Miller 1998b).
O O
N
H R
Strobel and coworkers (2002) isolated an endo-
Chaetoglobosin C R=O
Chaetoglobosin F R=OH phytic Pestalotiospis microspora from a small
tree stem of Terminalia morobensis collected on
the north coast of Papua New Guinea. Isopecta-
Me OH cin (Fig. 8.15), an isobenzofuranone, was
obtained from the culture broth of the fungal
Me
Me isolate (Strobel et al. 2002). The compound
H possessed antioxidant activity against a variety
Me of free radicals and a moderate antimycotic ac-
HN
O tivity against the plant pathogen Pythium ulti-
O O mum (Strobel et al. 2002).
N
H OH

Chaetoglobosin E
2. Sesquiterpenes from Pestalotiopsis sp.
from Pinus taeda
Pestalotiopsis sp. was the predominant endo-
O
phyte in a collection of microorganisms from
Me Pinus taeda from São Paulo State in Brazil
Me (Magnani et al. 2003). Three new highly oxidized
Me
caryophyllene sequiterpenes, named pestalotiop-
H
NH Me
solide A, taedolidol, and 6-epitaedolidol, were
HN purified from liquid culture of this endophytic
O
fungus (Fig. 8.15; Magnani et al. 2003). The car-
O
N
O
bon skeleton of pestalotiopsolide A is similar to
H
that of pestalotiopsin C (Magnani et al. 2003).
Phenochalasin A The pestalotiopsins A, B, and C were originally
described from endophytic Pestalotiopsis species
Fig. 8.14. Cytochalasan alkaloids from Chaetomium glo-
bosum from Imperata cylindrica isolated from Taxus brevifolia (Pulici et al. 1996,
1997). Pestalotiopsin A exhibited immunosup-
(Ding et al. 2006). The new cytotoxic cytochalasan- pressive and cytotoxic activity in preliminary
based alkaloid chaetoglobosin U and four known assays (Johnston et al. 2003). The pestalotiopsins
analogues chaetoglobosins C, F, E, and penochala- were not isolated by Magnani and coworkers,
sin A were isolated from exracts of the fungal but they assume that these compounds could be
biomass (Fig. 8.14; Ding et al. 2006). The cytotoxic precursors of pestalotiopsolide A, taedolidol, and
activity of all five compounds was tested against the 6-epitaedolidol.
 Endophytic Fungi, Occurrence and Metabolites 179

OH O

O OH
O
H

O
H O

HO O H

Isopectacin Pestalotiopsolide A

OH
OH O
O O
O

H
H O
O

Taedolidol 6-Epitaedolidol

O
OH O OH
O
HO O

O O
HO
O HO HO

HO HO

Pestafolide A Pestaphthalide A Pestaphthalide B

Fig. 8.15. Metabolites from endophytic Pestalotiopsis species

3. Metabolites from Pestalotiopsis foedan pestaphthalide A was active against C. albicans,

The endophytic fungus Pestalotiopsis foedan was
isolated from an unidentified tree (Ding et al.
2008). Bioassay-guided fractionation led to the
isolation of three new metabolites, a reduced
spiro azaphilone derivative named pestafolide
A, and the two isobenzofuranones pestaphtha-
lides A and B (Fig. 8.15; Ding et al. 2008). The
antifungal activity of the three compounds
was investigated against Candida albicans,
Geotrichum candidum, and Aspergillus fumiga-
tus in agar diffusion assays. Pestafolide A exhib-
ited antifungal activity against A. fumigatus,
and pestaphthalide B showed antifungal activity
against G. candidum (Ding et al. 2008).
4. Metabolites from Pestalotiopsis theae
The endophytic Pestalotiopsis theae was isolated
from an unidentified tree by Li et al. (2008). Four
new compounds, pestalotheols A–D (Fig. 8.16),
were purified from fungal rice cultures by bioas-
say-guided fractionation (Li et al. 2008). It is as-
sumed that the chromenones pestalotheols A, B,
and C are derived from two units of isoprenoids
and a polyketide, whereas pestalotheol C could be
180 Daniela Weber

R O
OH
O
OH
OH
O
O

O Pestalotheol A R=OH Pestalotheol C

Pestalotheol B R=H

O CH2OH

O O
OH
O

O
H
Pestalotheol D
(+)-(5S,10S)-4´-Hydroxy-
methylcyclozonarone

OH OH
O HO

O O
OH OH

R1
H H
R2
Tauranin R1=H2, R2=H Phyllospinarone H
3-Ketotauranin R1=O, R2=H R N O
3a-Hydroxytauranin R1=a-CH,b-H, R2=H
12-Hydroxytauranin R1=H2, R2=OH
O O O

O NH

H O N
H
O
O
O OH
Fusaristatin A R= NH2
O

OH
Fusaristatin B R=
CR377 O

Fig. 8.16. Metabolites from endophytic Pestalotiopsis theae, Phyllosticta spinarum from Platycladus orientalis, and
Fusarium sp.

their biosynthetic precursor. Of these four com-
pounds pestalotheol C showed an inhibitory effect
on HIV-1 replication in C8166 cells, but none of
the compounds exhibited noticeable antimicrobial
activity (Li et al. 2008).
J. Metabolites from Phyllosticta spinarum
from Platycladus orientalis
Platycladus orientalis (Cupressaceae) is a traditional Chi-
nese herb and food additive used for the treatment of gout,
 Endophytic Fungi, Occurrence and Metabolites
rheumatism, diarrhea, and chronic tracheitis (Lu et al. 2006).
The plant is also cultivated as an ornamental in southeastern
Arizona (Wijeratne et al. 2008).
The fungal endophyte Phyllosticta spinarum was
isolated from healthy foliage of Platycladus
orientalis collected in Arizona (Wijeratne et al.
2008). Five new sesquiterpene quinones and
related derivates, (+)-(5S,10S)-40 -hydroxymethyl-
cyclozonarone, 3-ketotauranin, 3a-hydroxytaura-
nin, 12-hydroxytauranin, and phyllospinarone,
together with tauranin were isolated from cultures
of P. spinarum (Fig. 8.16; Wijeratne et al. 2008).
The in vitro antiproliferative activity of the six
compounds was tested against five cell lines:
NCI-H460, MCF-7, SF-268, PC-3M, and MIA Pa
Ca-2. Only tauranin showed antiproliferative ac-
tivity against the cancer cell lines tested; all other
compounds were found to be inactive at concen-
trations up to 5 mM (Wijeratne et al. 2008).
K. Metabolites from Endophytic
Fusarium Species
1. CR377 from Fusarium sp. from
Selaginella pallescens
Species of the genus Selaginella are used in traditional Chi-
nese medicines, e.g. S. tamariscina and S. uncinata (Ma et al.
2003; Cheng et al. 2008). S. unicata is used to treat infectious
diseases and tumors and ethanol extracts of the whole plant
show significant inhibitory activities against respiratory
syncytial virus (Ma et al. 2003). S. tamariscina is a medicinal
plant used for the treatment of advanced cancer in the Orient
(Ahn et al. 2006) and for the therapy of chronic tracheitis
(Yang et al. 2007). The plant exhibits antitumor and vaso-
relaxant activity (Kang et al. 2004; Yang et al. 2007).
CR377, a Fusarium species, was collected in the
Guanacaste Conversation Area in Costa Rica and
isolated from a piece of a stem from another
Selaginella species, S. pallescens (Brady and
Clardy 2000). A new pentaketide, CR377
(Fig. 8.16), was isolated from the culture broth of
this Fusarium strain CR377. The compound
showed potent antifungal activity against strains
of Candida albicans in the agar diffusion assay
(Brady and Clardy 2000).
2. Lipopeptides from Fusarium sp. from Maackia
chinensis
Fungal strain Fusarium sp. YG-45 was isolated
from a twig segment of Maackia chinensis (Shiono
181
et al. 2007). The plant material was collected in the
botanical garden in Göttingen, Germany. From
fungal rice cultures two new cyclic lipopeptides,
fusaristatins A and B, were obtained (Fig. 8.16;
Shiono et al. 2007). These two compounds did
not exhibit antimicrobial activity at a concentra-
tion of 100 mg/ml against Staphylococcus aureus
NBRC 13276, Pseudomonas aeruginosa ATCC
15442, Candida albicans ATCC 2019, and Asper-
gillus clavatus F 318a (Shiono et al. 2007). Since
the fusaristatins are structurally related to
the DNA topoisomerase inhibitor, topostatin, the
inhibitory effect on topoisomerases was investi-
gated: fusaristatin B showed a moderate inhibition
of topoisomerase I and II with an IC50 value of 73
mM and 98 mM, respectively (Shiono et al 2007).
Fusaristatin A and B exhibited cytototoxic activity
against the human lung cancer cells LU 65 with an
IC50 value of 23 mM and 7 mM, respectively, where-
as no activity could be detected against the colon
cancer cells COLO 201 up to a concentration of
100 mM (Shiono et al. 2007).
L. Epichlicin from Epichloe typhina
from Phleum pretense L.
Infection by the choke disease endophytic fungus,
Epichloe typhina (Pers. Ex. Fr.) Tul., whose imma-
ture form is Acremonium typhinum, in Phleum
pretense induces resistance of leaf spot disease
caused by the pathogen, Cladosporium phlei
(Gregory) de Vries (Seto et al. 2007). In order to
explain the beneficial mutual relatioship between
fungus and host plant, Seto et al. (2007) investi-
gated the metabolites of E. typhina, since culture
broth of the endophytic fungus was able to inhibit
the spore germination in C. phlei. So they were
able to isolate the germination inhibitor epichlicin
(Fig. 8.17). This novel cyclic peptide with the
amino acid sequence 3-amino tetradecanoic acid
(b-amino acid), L-Asn, D-Tyr, L-Asn, L-Gln, L-
Ser, L-Asn, and D-Pro is a diastereomer of mixirin
A (Seto et al. 2007). Spore germination of C. phlei
was inhibited by epichlicin with an IC50 value of
22 nM (Seto et al. 2007).
M. Metabolite from Colletotrichum dematium
from Pteromischum sp.
Colletotrichum dematium was isolated from a
Pteromischum sp. growing in a tropical forest in
182 Daniela Weber

O
O HN (CH2)10CH3

HO O
NH HN
O O H2N O
NH NH2
N
H2N
NH O O
O O NH O
HN
OH
O
NH2

OH
Epichlicin H O
O
HO

H
HO

Brefeldin A
HO
OH OH
HO O O
OH O
O
O O O
O O
O

Hormonemate O O
N N
O O
O O

OH OH
HO HO

Phaeosphaeride A Phaeosphaeride B

Fig. 8.17. Structures of epichlicin, brefeldin A, hormonemate, and phaeospaerides A and B

Costa Rica (Ren et al. 2008). The fungus pro- cyclosporine A in the same test yielded a value
duced a novel antimycotic peptide, colutellin A, of 61.8 nM (Ren et al. 2008).
which contains residues of Ile, Var, Ser, N-meth-
yl-Val, and beta-aminoisobutryic acid in nomi-
nal molar ratios of 3:2:1:1:1 (Ren et al. 2008). N. Hormonemate from Hormonema
Colutellin A is assumed to have potential im- dematioides from Pinus sp.
munosuppressive activity, since it inhibited
CD4(+) T-cell activation of interleukin 2 produc- The fungal strain E99156 was isolated as a symp-
tion with an IC50 value of 167.3 nM, wherease tomless endophyte from the needle base of a Pinus
 Endophytic Fungi, Occurrence and Metabolites
species collected in Portugal (Filip et al. 2003). The
endophyte was identified as Hormonema dema-
tioides by microscopic and genetic characteristics.
The new metabolite hormonemate was isolated
from submerged cultures of the endophytic fun-
gus (Fig. 8.17; Filip et al. 2003). The compound
exhibited cytotoxic acitivity against the tested
cancer cell lines COLO 320, DLD-1, HT-29, Jurkat,
and HL-60 cells, with IC50 values of 3.5 mg/ml and
3.75 mg/ml, and induced apoptosis in COLO-320
cells (Filip et al. 2003).
O. Brefeldin A from Phoma medicaginis
from Medicago lupulina
Phoma medicaginis was the dominating endophyte
in Medicago sativa and M. lupulina (Weber et al.
2004). The macrocyclic lactone brefeldin A
(Fig. 8.17), an inhibitor of protein trafficking in
the endomembrane system of mammalian cells
(Nebenführ et al. 2002), was isolated from pure
fungal cultures and detected in infected and artifi-
cially inoculated plant material of M. sativa (Weber
et al. 2004). Brefeldin A was not found in freshly
harvested plant material, so the authors assumed
that this secondary metabolite is produced to de-
fend the dead plant against other saprophytic
organisms after the switch from the endophytic to
the saprotrophic phase, following the death of
infected host tissue (Weber et al. 2004).
P. Phaeosphaerides from an
Endophytic Fungus
Maloney and coworkers (2006) isolated endophyt-
ic fungi from plant samples collected in the Arch-
bold Biological Station, a 5000-acre (20.2 km2)
preserve in Lake Placid in Florida. The endophyte
FA 39, having 97% identity to the ascomycete
Phaeosphaeria avenaria, exhibited interesting ac-
tivity against STAT3 (Maloney et al. 2006). Two
compounds, phaeosphaeride A and B, were pu-
rified from fungal material and have a new carbon
skeleton related to the fungal curvupallides
and the lipid-lowering spirostaphylotrichins/
triticones (Fig. 8.17; Maloney et al. 2006). These
three families share the functional group O-meth-
yl hydroxamic acid (Maloney et al. 2006). In
an ELISA-based screening phaeosphaeride A
183
inhibited STAT3, with an IC50 value of 0.61 mM,
whereas the diastereomer, phaeosphaerid B,
exhibited no activity against STAT3 (Manoley
et al. 2006). Phaeosphaeride A is an inhibitor of
STAT3 signaling and selective for STAT3 over
other STAT proteins (Manoley et al. 2006).
Q. Metabolites from Nodulisporium sp. from
Junipercus cedre
The fungus Nodulisporium sp. 7080, belonging to
the Xylariaceae, was isolated from twigs of the plant
Juniperus cedre (Dai et al. 2006). Searching for
biologically active compounds from endophytic
fungi, Dai and coworkers (2006) found that the
crude extracts of Nodulisporium sp. 7080 exhibited
antimicrobial and algicidal activities. Seven new
natural products, 3-hydroxy-1-(2,6-dihydroxyphe-
nyl)-butan-1-one, 1-(2-hydroxy-6-methoxyphenyl)
butan-1-one, 2,3-dihydro-5-methoxy-2-methyl-
chomen-4-one, the dimeric naphthalenes noduli-
sporin A and B, the first naturally occurring
dimeric indanone nodulisporin C, and (4E,6E)-
2,4,6-trimethylocta-4,6-dien-3-one, and ten known
compounds were isolated by bioassay guided
fractionation (Fig. 8.18; Dai et al. 2006). The
known compounds were identified by NMR
spectroscopy as 1-(2,6-dihydroxyphenyl)butan-
1-one, 5-hydroxy-2-methyl-4H-chromen-4-one,
2,3-dihydro-5-hydroxy-2-methylchomen-4-one, 8-
methoxynaphthalen-1-ol, 1,8-dimethoxynaphtha-
lene, daldinol, helicascolide A, the eudesmane
derivative (1S,4S,4aS,6R,6aR)-decahydro-1,4-di-
methyl-6-(prop-1-en-2-yl)naphthalene-1-ol, ergos-
terol, and 5a,8a-epidioxyergosterol (Fig. 8.18, 8.19;
Dai et al. 2006). 1-(2-Hydroxy-6-methoxyphenyl)
butan-1-one and 1-(2,6-dihydroxyphenyl)butan-1-
one exhibited biological activity at a concentration
of 0.25 mg/filter in the agar diffusion assay against
the fungi Microbotryum violaceum and Septoria
tritici and the green alga Chlorella fusca (Dai et al.
2006).
R. Metabolites from Ascochyta sp. from
Meliotus dentatus
The endophytic fungus Ascochyta sp. 6651 was
isolated from the plant Meliotus dentatus (Krohn
et al. 2007b). The plant material was collected at the
184 Daniela Weber

OH O
Fig. 8.18. Metabolites from Nodu- O
lisporium sp. from Junipercus
cedre – part 1
OH O OH O O
OH O

3-Hydroxy-1-(2,6-di- 1-(2-Hydroxy-6-methoxy- 2,3-Dihydro-5-methoxy-

hydroxyphenyl)-butan-1-one phenyl)butan-1-one 2-methylchromen-4-one

OH
O OH
(4E,6E)-2,4,6-Trimethyl-
octa-4,6-dien-3-one

O OH
Nodulisporin A

OH O
O OH
O
Nodulisporin B

OH

OH O O
1-(2,6-Dihydroxyphenyl)- HO O
butan-1-one
Nodulisporin C

O O

OH O OH O OH O

5-Hydroxy-2-methyl- 2,3-Dihydro-5-hydroxy-
4H-chromen-4-one 2-methylchromen-4-one 8-Methoxynaphthalene

shores of the Baltic Sea. Two new compounds, (4S)-
(+)-ascochin and (S,S)-(+)-ascodiketone, and three
known compounds, (3R,4R)-()-4-hydroxymel-
lein, ent-a-cyperone, and (3S,4R)-()-dihydroxy-
(6S)-undecyl-a-pyranone, were purified from
fungal cultures of Ascochyta sp. 6651 (Fig. 8.20;
Krohn et al. 2007b). Ascochin, 4-hydroxymellein,
and dihydroxy-undecyl-pyranone showed activity
against the gram-positive bacterium Bacillus mega-
sterium, the fungus Microbotryum violaceum, and
the alga Chlorella fusca in the agar diffusion test at a
concentration of 0.5 mg/filter (Krohn et al. 2007b).
S. Metabolites from Endophytic Fungi from
Garcinia Species
Species of the genus Garcinia are used in ethnopharma-
cology, e.g. Garcinia mangostana L. (mangosteen) is a tree
widespread in Southeast Asian countries with medicinal
properties (Suksamrarn et al. 2003). The fruit hulls are
 Endophytic Fungi, Occurrence and Metabolites 185

HO

O
O
O O
OH

1,8-Dimethoxynaphthalene Daldinol

O
OH
H
O

OH
H

(1S,4S,4aS,6R,8aR)-Deca-
hydro-1,4-dimethyl-6-(prop- Helicascolide A
1-en-2-yl)-naphthalen-1-ol

O O
HO
HO

Ergosterol 5a,8a-Epidioxyergosterol

Fig. 8.19. Metabolites from Nodulisporium sp. from Junipercus cedre – part 2

used in Thai folk medicine for the treatment of skin infec-
tions, wounds, and diarrhea (Suksamrarn et al. 2003) and
as an anti-inflammatory drug (Chen et al. 2008). Pedraza-
Chaverri et al. (2008) reported that the pericarp (peel, rind,
hull, or ripe) of G. mangostana is used in traditional
medicine for the treatment of abdominal pain, diarrhea,
dysentery, infected wound, suppuration, and chronic
ulcer. Experimental studies demonstrated that extracts of
the plant have antioxidant, antitumoral, antiallergic, anti-
inflammatory, antibacterial, and antiviral activities (Ped-
raza-Chaverri et al. 2008).
The endophytic fungus Botryosphaeria mamane
PSU-M76 was isolated from the leaves of
G. mangostana collected in Thailand (Pongchar-
oen et al. 2007). A new dihydrobenzofuran,
botryomaman, was isolated along with six
known compounds, 2,4-dimethoxy-6-pentylphe-
nol, (R)-()-mellein, primin, cis-4-hydroxymel-
lein, trans-4-hydroxymellein, and 4,5-dihydroxy-
2-hexenoic acid (Figs. 8.20, 8.21 Pongcharoen
et al. 2007). Pongcharoen and coworkers (2007)
assumed that botryomaman is derived from the
condensation of the demethylated 2,4-dimethoxy-
6-pentylphenol and 4,5-dihydroxy-2-hexenoic
acid. Primin exhibited the best activity against
186 Daniela Weber

O
OH
HO O O

O (CH2)11 O

OH O OH O

(S)-(+)-Ascochin (S,S)-(+)-Ascodiketone (3R,4R)-(-)-4-Hydroxymellein

OH
HO

O (CH2)9
O O

ent-(-)-a-Cyperone (3S,4R)-(-)-Dihydroxy-(6S)-undecyl-a-pyranone

HO
O
H
HO HO
H

O O O
O

Botryomaman 2,4-Dimethoxy-6-pentyl phenol

O OH O

O
O
H

R2 R1
O

(R)-(-)-Mellein R1=R2=H
cis-4-Hydroxymellein R1=H, R2=OH
Primin trans -4-Hydroxymellein R1=OH, R2=H

Fig. 8.20. Metabolites from Ascochyta sp. from Meliotus dentatus and from endophytic fungi from Garcinia species

Staphylococcus aureus and methicillin-resitant S.
aureus, with MIC values of 8 mg/ml, while all the
other compounds were inactive, with MIC values
>128 mg/ml (Pongcharoen et al. 2007).
The endophyte Penicillium paxilli PSU-A71 was
isolated from leaves of G. atroviridis collected in
Thailand (Rukachaisirikul et al. 2007). A new pyrone
derivative, penicillone, together with paxilline and
pyrenocines A and B were obtained from extracts
of fungal fermentations (Fig. 8.21; Rukachaisirikul
et al. 2007). Pyrenocine B showed mild antifungal
activity against the human pathogenic fungus Micro-
sporum gypseum with a MIC value of 32 mg/ml,
whereas penicillone and pyrenocine A were much
less active with MIC values of 64 mg/ml and 128 mg/
ml, respectively (Rukachaisirikul et al. 2007).
 Endophytic Fungi, Occurrence and Metabolites 187

SH
O

O
OH

HO
O

HO O O

4,5-Dihydroxy-2-hexenoic acid Penicillone

H
O
OH
O O

N OH
H O O
O
H H

Paxilline Pyrenocine A

O
OH O
O OH
H
O
H

O O
H

Pyrenocine B Leptosphaeric acid

OH OH

OH OH
O
O
HO HO
H

O O
H H H H

Daldinone C Daldinone D

Fig. 8.21. Metabolites from endophytic fungi from Garcinia and Artemisia species

T. Metabolites from Endophytic Fungi from
Artemisia species
Artemisia species are widespread in nature and are fre-
quently used for the treatment of malaria, hepatitis, can-
cer, inflammation, and infections by fungi bacteria and
viruses (Tan et al. 1998). The endoperoxides sequiterpene
lactone artemisinin was isolated from A. annua. The high-
est artemisinin concentrations can be found in the leaves
and inflorescences (Woerdenbag et al. 1990). Artemisinin
and its derivatives are a potent new class of antimalarials
(Balint 2001), since artemisinin is effective against both
drug-resistant and cerebral malaria caused by strains of
Plasmodium falciparum (Abdin et al. 2003).
The endophyte Leptosphaeria sp. IV403 was isolated
from the plant Artemisia annua. Liu and coworkers
(2003) purified a metabolite, leptosphaeric acid
188 Daniela Weber

OH O

OH O OH O

(4S)-5,8-dihydroxy-4-
Altechromone A
methoxy-a-tetralone

OH O O
O
O
O
O
O OH
HO HO
O
OH O

Colletotric acid Massarilactone E

O
O
O
O
O
O
O
O
O
O O
O
HO O
HO
O
O

Massarilactone F Massarilactone G Massarilactone acetonide

O O

O O
OH

O OH O O

DWE01141-1 2,7-Dimethoxy-6-(1-acetoxyethyl)-juglon

Fig. 8.22. Metabolites from endophytic fungi from Artemisia species and Schinus molle

(Fig. 8.21), with a novel carbon skeleton and unre-
lated to artemisin from extracts of the culture of the
endophyte.
The endophytic Hypoxylon truncatum IFB-18
was isolated from symtomless stem tissue of A.
annua by Gu et al. (2007). Two new benzo[j]fluor-
anthene-based natural products, daldinone C and
daldinone D, and the known metabolites altechro-
mone A and (4S)-5,8-dihydroxy-4-methoxy-a-tet-
ralone were isolated from extracts of a solide
culture of the fungal strain (Figs. 8.21, 8.22; Gu
et al. 2007). Daldinones C and D exhibited cyto-
toxic ativity against the SW1116 cell line with IC50
values of 49.5 mM and 41.0 mM (Gu et al. 2007).
 Endophytic Fungi, Occurrence and Metabolites
Biosynthetically these compounds could be
derived from a polyketide precusor.
Artemisia mongolica is known to be strongly
resistant to insects and pathogens (Zou et al. 2000).
From apparently healthy plants the fungus
Colletotrichum gloeosporioides was isolated (Zou
et al. 2000). From the culture liquid of this fungus
a new metabolite, named colletotric acid (Fig. 8.22),
was isolated. C. gloeosporioides is known as a host-
specific pathogen causing anthracnose of host
plants and some strains are of value for biological
control of weeds (Zou et al. 2000). Antimicrobial
bioassays revealed that colletotric acid was anti-
bacterial to Bacillus subtilis, Staphylococcus aureus,
and Sarcina lutea, with MICs of 25, 50, and 50 mg/
ml and antifungal to Helmithosporium sativum
with a MIC of 50 mg/ml (Zou et al. 2000).
The fungal isolate 7303, a Coniothyrium
species, was isolated from the halotolerant plant
Artemisia maritima (Krohn et al. 2007a). The
plant material was collected from Mandø in Den-
mark. Three new metabolites, massarilactones E-
G and massarilactone acetonide (Fig. 8.22), named
according to related metabolites from the fresh-
water fungus Massarina tunicata, were isolated
from extracts of fungal cultures (Krohn et al.
2007a).
U. Metabolites from Endophytes from
Schinus molle
The genus Schinus comprises 30 species, which are origi-
nally widespread from Mexico to Argentina (Roth et al.
1994). S. molle is used in ethnopharmacology as a vulner-
ary and anti-inflammatory agent (Quiroga et al. 2001) and
the fruits are used as spice. Extracts of S. molle showed
antifungal activities against several fungi (Quiroga et al.
2001). The oil of S. molle exhibited fungitoxic activity
against some common storage and animal pathogenic
fungi (Dikshit et al. 1986).
Artemisia new compound, DW E01141-1, was
isolated from an endophytic Colletotrichum
gloeosporioides, isolated from leaves of S. molle
(Fig. 8.22; Weber 2006). 2,7-Dimethoxy-6-(1-acet-
oxyethyl)-juglon was purified from Botryo-
sphaeria parva, which was isolated from small
woody twigs of S. molle (Weber 2006). DW
E01141-1 exhibited no antimicrobial, cytotoxic,
phytotoxic, or nematicidal activities (Weber
2006). 2,7-Dimethoxy-6-(1-acetoxyethyl)-juglon
189
is an orange pigment described from Henderso-
nula toruloidea (Fig. 8.22; van Eijik and
Roeymans 1978) and Kirschsteiniothelia sp.
(Poch et al. 1992).
V. Conclusion
Endophytic fungi are still an interesting source for
new natural products. New metabolites together
with already known compounds are isolated from
cultures of endophytic fungi. This confirms the
hypothesis of Seymour et al. (2004) that endo-
phytic isolates which are genotypically similar
show considerable metabolite variability. The as-
sessment of the potential metabolic diversity of a
fungal species cannot be adequately performed by
investigating a single ‘‘representative’’ isolate
(Seymour et al. 2004). Fungal species possess an
interesting potential for secondary metabolic var-
iation.
There is an increasing number of reports of
endophytic fungi that produce metabolites origi-
nally known from their host plants. This was first
described for taxol produced by Pestalotiopsis
andreanae and Taxus brevifolia. It can be as-
sumed that there occurred a horizontal gene
transfer, but the details of this gene transfer are
still unknown. These new publications support
the hypothesis that endophytic fungi adapt to the
microenvironment in the host plants and are able
to uptake plant DNA into their genome.
The isolation of endophytes from plants is
described in various publications. In the past
years many authors reported in detail the isolation
of endophytic fungal communities from host
plants (Kumar and Hyde et al. 2004; Nalini et al.
2005; Raviraja 2005; Tejesvi et al. 2005; Gond et al.
2007; El-Zayat et al. 2008) or the screening of
crude extracts of endophytic fungi for bioactivity
(Wiyakrutta et al. 2004; Li et al. 2005). Since it is
often mentioned that the isolation is performed in
order to investigate the secondary metabolites of
the endophytic fungi, it can be expected that the
literature on natural compounds from endophytes
will increase constantly in the next years.
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9 Fungal Origin of Ergoline Alkaloids Present in Dicotyledonous
Plants (Convolvulaceae)

ECKHARD LEISTNER1, ULRIKE STEINER2

CONTENTS which are also called secondary metabolites – are

I. The Ecological Role of Natural Products . . . . . . 197 characteristic of a limited number of microbial or
II. The Symbiosis Between Poaceae and plant taxa, e.g. an order, a family, a species or even
Clavicipitaceous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . 198 a subspecies only. Many of the natural products
III. Epibiotic Clavicipitaceous Fungi Associated exhibit physiological activities which is the basis
with Convolvulaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
A. Identification of Clavicipitaceous Fungi . . . . 199 for their use in medical applications (Clardy and
1. Microscopic and Electron Walsh 2004).
Microscopic Characterisation . . . . . . . . . 199 The high physiological activities of many nat-
2. Phylogenetic Trees . . . . . . . . . . . . . . . . . . . . . 202 ural products triggered a now historical dispute
B. Fungicidal Treatment . . . . . . . . . . . . . . . . . . . . . . . 202 about the role of natural products in the produc-
C. Plant Growth Under Germ-Free
Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 ing organism. It was proposed that ‘‘the multiplic-
D. Biosynthesis and Accumulation of ity of natural products is caused by random
Ergoline Alkaloids in the Fungus/Plant processes of mutations, i.e. it reflects the gambling
Symbiotum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 of nature rather than a sophisticated strategy’’
E. Seed Transmittance of Epibiotic Fungi (Mothes 1981; Mothes et al. 1985).
Colonizing Convolvulaceae . . . . . . . . . . . . . . . . . 204
IV. Additional Fungus/Plant Symbiota in This hypothesis, however, neglects the possi-
Dicotyledonous Plants . . . . . . . . . . . . . . . . . . . . . . . . . . 205 bility that mutations may turn out to be detrimen-
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 tal or advantageous to the mutated organism. In
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 the former case a mutated organism may be elimi-
nated, or in the latter case may benefit from an
increased fitness and a better chance to survive in
a certain ecological setting (Zenk 1967). Today the
Dedicated to Prof. Dr. Detlef Gröger on the occa- ecological role of natural products is well accepted
sion of his 80th birthday in the scientific community (Eisner 2003; White
Jr. et al. 2003; Harborne 2004).
Natural product research entered a new era
I. The Ecological Role of Natural when it was discovered that plants and fungi ela-
Products borated during evolution another way to acquire
natural products: Not only may they be formed in
biosynthetic processes by one particular organism
Microorganisms and plants have one thing in
itself, but a host organism may instead harbor a
common: both are equipped with a frequently
natural product-producing microorganism: A
elaborate biosynthetic machinery responsible for
plant may be associated with a bacterium (Piel
the formation of an almost unlimited variety of
2004; Strobel 2004; Gunatilaka 2006) or a fungus
natural products. Typically, natural products –
(Strobel 2004; Puri et al. 2005; Gunatilaka 2006),
while a fungus may harbor a bacterium (Partida-
1
Institut für Pharmazeutische Biologie, Rheinische Friedrich Martinez and Hertweck 2005).
Wilhelm-Universität Bonn, Nussallee 6, 53115 Bonn, Germany;
It is remarkable that three important and fre-
e-mail: eleistner@uni-bonn.de
2
Institut für Nutzpflanzenwissenschaften und Ressourcenschutz
quently used cytostatic compounds employed in
(INRES), Rheinische Friedrich Wilhelm-Universität Bonn, Nus- today’s tumor therapy apparently are synthesized
sallee 9, 53115 Bonn, Germany; e-mail: u-steiner@uni-bonn.de by plant-associated microorganisms: Vincristine

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
198 Eckhard Leistner and Ulrike Steiner
is produced by Fusarium oxysporum associated
with Catharanthus roseus (Zhang et al. 2000;
cited by Gunatilaka 2006), camptothecin, a lead
compound in cancer research and therapy is pro-
duced by a fungal endophyte present in Nothapo-
dytes foetida (Puri et al. 2005) and paclitaxel is
believed to be formed by different fungi such as
Taxomyces andreanae and different Taxus spe-
cies, including T. brevifolia (Strobel et al. 2004;
Leistner 2005). The latter case, however, may not
yet be setteled because the genes in Taxomyces
andreanae have not yet been reported whereas
they are well known from the Taxus brevifolia
plant (Croteau et al. 2006).
At present it is unclear whether these natural
products and their biosynthetic machinery occur
exclusively in the associated microorganism or in
both the microorganism and the host plant. In
such associations both organisms may be part of
a symbiotum in which the associated microorgan-
ism benefits by receiving nutrients, protection,
reproduction and dissemination, whereas the
host takes advantage of physiologically active
compounds which may promote plant growth,
herbivore deterrence and/or increased fitness
(Arnold et al. 2003; White Jr. et al. 2003; Saikko-
nen et al. 2004).
A point in case is the beneficial activity of
ergoline alkaloids which are products of clavicipi-
taceous fungi (Clay and Schardl 2002; White Jr.
et al. 2003; Schardl et al. 2006) colonizing grasses
like Poaceae, Juncaceae and Cyperaceae.
In the current literature (including our own
publications) two different adjectives for the
fungi described herein are used: These are ‘‘cla-
vicipitaceous’’ and ‘‘clavicipitalean’’. The former
term refers to the family Clavicipitaceae but the
latter to the old order of Clavicipitales. Since the
family Clavicipitaceae is now generally accepted
to belong to the order Hypocreales (Sung et al.
2007) only the term ‘‘clavicipitaceous’’ should
be used. We are grateful to Dr. Chris Schardl
(Lexington, Kentucky, USA) for bringing this to
our attention.
Ergoline alkaloids, however, are also present
in higher dicotyledonous plants of the family Con-
volvulaceae (Hofmann 1961, 2006). This disjoint-
ed occurrence of a group of natural products in
evolutionarily unrelated taxa (fungi, Convolvula-
ceae plants) seemed to contradict the generally
accepted principle of chemotaxonomy that similar
or even identical natural products are present in
related taxa. It was therefore assumed that during
evolution a horizontal transfer of genes responsi-
ble for ergoline alkaloid biosynthesis might have
occurred from fungi to higher plants (Groeger and
Floss 1998; Tudzynski et al. 2001; Clay and Schardl
2002). Alternatively it was discussed that ergoline
alkaloid biosynthesis was repeatedly invented
during evolution (Mothes et al. 1985). In a recent
review in this series Keller and Tudzinsky (2002)
dealt with the pharmacological aspects, biochem-
istry, genetics and biotechnology of ergoline alka-
loids in fungi associated with Poaceae. We show in
the present review that neither the horizontal
transfer of genes encoding the ergoline alkaloid
biosynthesis, nor the repeated invention of a rather
complicated biosynthetic pathway took place dur-
ing evolution, but rather that clavicipitaceous fungi
not only live on different grasses but also colonize
plant species of the dicotyledonous family of Con-
volvulaceae (Kucht et al. 2004; Steiner et al. 2006;
Ahimsa-Mueller et al. 2007; Markert et al. 2008;
Steiner et al. 2008). This indicates that ergoline
alkaloids are components in a fungus/plant symbio-
tum characterized by mutual defense.
II. The Symbiosis Between Poaceae
and Clavicipitaceous Fungi
A rather well investigated experimental system
consists of clavicipitaceous fungi which colonize
Juncaceae, Cyperaceae and Poaceae plants. In
these symbiota ergoline alkaloids play an impor-
tant role (Keller and Tudzynski 2002). The symbi-
otic fungi belong either to the tribe Clavicipeae or
Balanseae within the family Clavicipitaceae (Bacon
and Lyons 2005). The morphological associations
of the fungi with grasses is either epicuticular,
epibiotic or endophytic (Bacon and Lyons 2005).
In epiphytic growth the fungal mycelium is con-
centrated on the surface of young leaves, buds,
meristematic regions and reproductive structures
(Clay and Schardl 2002). The association between
fungi and their plant hosts is likely to be an exam-
ple of host-symbiont codivergence (Schardl et al.
2008).
The fungus may be asexual belonging to the
group of fungi imperfecti, show a sexual lifestyle or
switch between sexual and asexual propagation. In
the sexual lifestyle fungi parasitize a wide range of
grasses where they form infections of single grass
 Fungal Origin of Ergoline Alkaloids Present in Dicotyledonous Plants (Convolvulaceae)
florets and replace the seed with individual scler-
otia (Clay and Schardl 2002).
The asexual fungi are vertically transmitted
through seeds. They have never been known to
produce infectious spores and rely entirely on seed
transmission. Especially the asexual fungi exhibit
high host specificity. Most interestingly, sexual and
asexual fungi may interact in parasexual processes
contributing to a high diversity of fungal asexual
endophytes (Tsai et al. 1994).
In general, grasses are poor producers of natu-
ral products that assist other plants in their long-
term strategy to gain an ecological advantage.
Grasses, however, have the ability to compensate
for this deficiency by acquiring fungi notorious for
their poisonous natural products. In some cases
fungi can be considered the lifestock of grasses.
Fungi associated with plants may produce dif-
ferent classes of alkaloids among which toxic
ergoline alkaloids are an important group
(Schardl et al. 2004, 2007). The main ecological
roles of ergoline alkaloids in nature are probably
to protect the fungi from consumption by verte-
brate and invertebrate animals (Schardl et al.
2006). Ergoline alkaloids benefit the fungus by
protecting the health and productivity of the
host (Schardl et al. 2006). Other benefits include
growth of the plant, competitive abilities, resis-
tence to drought (Malinowski and Belesky 2000),
pests and fungal pathogens (Brem and Leucht-
mann 2002; White Jr. et al. 2003). In some cases
clavicipitaceous fungi are culturable in vitro
(Keller and Tudzynski 2002). This helped to iden-
tify the fungus as the producer of ergoline alka-
loids and revealed that the host plant is not the site
of ergoline alkaloid biosynthesis.
It was therefore somewhat unexpected when
Hofmann (1961, 2006) found that dicotyledonous
plants belonging to the family Convolvulaceae
contained ergoline alkaloids and that these alka-
loids were responsible for the hallucinogenic
properties enjoyed by Meso- and South American
indians in religious ceremonies.
The idea that a fungus could be responsible
for the alkaloid occurrence was discussed but no
evidence for the presence of such a fungus was
found (Hofmann 2006). This seemed to be in
agreement with the notion that plant tissue cul-
tures which are believed to be germ-free, i.e.
devoid of any microbes, were reported to produce
ergoline alkaloids (Dobberstein and Staba 1969;
see below).
199
III. Epibiotic Clavicipitaceous Fungi
Associated with Convolvulaceae
A. Identification of Clavicipitaceous Fungi
1. Microscopic and Electron Microscopic
Characterization
The infestation of the clavicipitaceous fungi
on members of the family Convolvulaceae is sys-
temic. Evidence of systemic infection came from
demonstrations that the fungi are seed transmit-
ted, that surface-sterilized seeds grown in vitro
and under germ-free conditions result in plantlets
which are colonized exclusively by the respective
clavicipitaceous fungi and that they are transmit-
ted through vegetative propagation (Steiner et al.
2008). It is an unusual type of systemic infection
in that there are no signs of penetration into the
host tissue and growth on the host plant is super-
ficial. Attempts made to visualize the fungus with-
in the stem and leaf tissue, using methodologies
commonly employed to detect endophytes in
grasses (Bacon and White Jr. 1994), were not suc-
cessful. Up to now the fungi have proved to be
non-detectable using these procedures. Among
the Clavicipitaceae, Atkinsonella hypoxylon,
Balansia cyperi, B. pilulaeformis and Myriogenos-
pora atramentosa are examples of epibiotic spe-
cies that grow on the meristematic tissues of host
plants (Luttrell and Bacon 1977; Rykard et al.
1985; Leuchtmann and Clay 1988, 1989; Clay and
Frentz 1993). The clavicipitaceous fungi coloniz-
ing members of the Convolvulaceae inhabit an
epibiotic niche and thus seem most comparable
to the epibiotic members of the grass borne Cla-
vicipitaceae. The mutualistic endophyte Neothy-
phodium typhinum also forms a stable external
mycelial net on the leaves of the host plant (Moy
et al. 2000). This suggested a possible alternative
pathway of fungal dispersal and transmission to
hosts, i.e. through epiphyllously produced conidia.
The clavicipitaceous fungi form colonies on
the upper surfaces of young unfolded leaves
which are visible to the naked eye, as shown
for Turbina corymbosa (Fig. 9.1A) and Ipomoea
asarifolia. They are also detectable by molecular
biological techniques in seeds of I. violacea
(Ahimsa-Müller et al. 2007). On T. corymbosa
colony distribution mainly follows the veins
of the leaves (Fig. 9.1A, B), in contrast to the
distribution on I. asarifolia which is more
200 Eckhard Leistner and Ulrike Steiner

A B
gsc

mm

500 µm
C
D

ml

hy

gsc
20 µm c

E hy F

hy
gsc

hy scc c
hy
sc

bc
gsc
500 µm 10 µm 1 µm

Fig. 9.1. Colonization of Turbina corymbosa with the
clavicipitaceous fungus (provisionally named TcorF01).
A Colonies formed by white mycelium on the adaxial
surface of a young unfolded leaf. Preferential development
on the veins is visible with the naked eye. B Aggregated
hyphae differentiating typical mycelium mats (mm) con-
sisting of several layers which cover leaf areas with peltate
glandular trichomes and are adhered to the cuticle. C
Cross-section of a closed leaf bud showing that the fungus
is well established on the adaxial leaf surfaces at this early
stage of plant development. The mycelium is formed by
tightly packed hyphae as a mycelium layer (ml, arrows) in
the cavity between the halves of the leaf. D Close associa-
tion of secretory cells (gsc) on the adaxial leaf surface with
hyphae (hy) which often encircle the peltate glandular
trichomes of the plants. E Cross-section of a peltate glan-
dular trichome composed of basal cell (bc), stalk cell (sc)
and secretory cells (gsc) showing the epiphytic develop-
ment of mycelium embedded in a mucilage matrix con-
centrated on the cuticle over a subcuticular oil storage
cavity. F Electron microscopic view of secretory cells
with hyphae outside and inside of the subcuticular oil
storage cavity (scc) bordered by the cuticle (c, arrow). No
evidence for direct penetration of the plant cells is visible
 Fungal Origin of Ergoline Alkaloids Present in Dicotyledonous Plants (Convolvulaceae)
random. These colonies differ in size and myceli-
um density, and depending on the developmental
stage, the fungi produce synnemata-like struc-
tures. No stromata with perithecia and ascospores
were detected in the mycelium mats. Maybe the
environmental conditions are not suitable for the
development of the sexual stage of the fungi, or
they lost the ability to reproduce sexually, or the
mating type is lacking. No traces of mycelium
were detected on the lower side of the leaves.
Visual inspection of leaf buds which were opened
by manipulation showed that the fungus was well
established as dense white mycelial layers on the
adaxial leaf surfaces of both I. asarifolia and
T. corymbosa plants (Fig. 9.1C) at this early stage
of plant development. The mycelium is formed by
tightly packed hyphae in the cavity between the
leaf halves. Sections through colonized tissue
revealed that the fungal mycelium is entirely
superficial. The hyphae measured approximately
1.0–1.5 mm across, were hyaline, thin-walled
and septated. Chlamydospore-like structures are
produced. As indicated by the intense mycelium
development, the space between the upper sur-
faces of folded leaves probably offer a refuge of
protection to the fungus. As leaves expand and
mature the hyphae are evident as isolated clumps
only visible microscopically, often near or around
peltate glandular trichomes, and the ends of the
hyphae often appeared broken (Fig. 9.1D).
The epibiotic fungi of I. asarifolia and T. corym-
bosa (Fig. 9.1D, E) are closely associated with secre-
tory glands on the adaxial leaf surface, an anatomic
feature which may be essential for ergoline alkaloid
biosynthesis in the epibiotic fungus/plant associa-
tion (Steiner et al. 2008). In cell cultures which har-
bor the fungus no ergoline alkaloids are synthesized
and no secretory glands are developed.
Members of the Convolvulaceae like I. asar-
ifolia and T. corymbosa (Fig. 9.1E) form peltate
glandular trichomes, which consist of one basal
cell, one stalk cell, up to eight glandular secretory
cells and a subcuticular oil storage cavity that is
derived from the cuticle of the secretory cells.
Metabolites are released after rupture of the cu-
ticle. As indicated by staining with the lipophilic
dye Nile red these specialized structures contain
essential oils (Kucht et al. 2004). The secretory
glands and their specific metabolites may be the
basis of a metabolic dialog between fungus and
plant (Steiner et al. 2008). The fungi may feed
on the volatile oil and derive precursors like
201
terpenes for ergoline alkaloid biosynthesis from
the oil. The fungi inhabit the epibiotic niche of
glandular cells on the upper surface of leaves. This
observation is supported by showing hyphae of
the clavicipitaceous fungus on T. corymbosa both
outside of the subcuticular oil storage cavity and
inside of this compartment embedded in an elec-
tron-dense matrix (Fig. 9.1F). The localization of
mycelium with the glandular cells ensures the
close association between fungus and host tissues.
A continuous maintenance of the symbiotic rela-
tionship requires that the fungus derives energy
from the host plant. In clavicipitaceous epibiotic
fungi, substrate utilization depends on the avail-
ability of organic material from the waxy cuticle
covering the plant surface and exuded com-
pounds, lipids, amino acids and vitamins. The
main energy-yielding compounds are simple
sugars that in the case of endophytic mycelia are
derived from the apoplasm through intercellular
fungal hyphae (White and Morgan-Jones 1996). In
clavicipitaceous fungi present on I. asarifolia and
T. corymbosa, superficial fungal hyphae have been
observed with tip enlargements tightly adherent
to the glandular cells as well as to the cuticle.
Probably, mechanisms for a selective and efficient
transfer of carbohydrates to the fungus could be
present.
Physiological changes paralleled by morpho-
logical adaptations of the host have been de-
scribed for some endophytic associations (Bacon
and White Jr. 2000). In M. atramentosa, plant host
changes in the epidermal cell size and shape sug-
gest the activity of growth regulatory substances
which are either produced by the fungus or secret-
ed into the host or are produced by the host in
response to the fungal symbiont (Bacon and
White Jr. 2000).
The epiphytic proliferation of hyphae on the
cuticle may be additionally enabled through deg-
radation of the cuticular layers of the leaf surfaces.
Previous ultrastructural studies of the host–
fungus interfaces of the clavicipitaceous fungi on
I. asarifolia and T. corymbosa revealed progres-
sive cuticular disintegration. Substrate utilization
studies have shown that epiphytic members of the
Balansiaceae such as Atkinsonella hypoxylon, pos-
sess the capacity to colonize and degrade paraffin
wax droplets (White Jr. et al. 1991). A. hypoxylon
grows superficially on young leaves of grasses as
an epiphyte, perhaps degrading wax in the cuticle
to obtain nutrients for epiphytic growth (White Jr.
202 Eckhard Leistner and Ulrike Steiner
et al. 1991). Leaves and inflorescence primordia
within the stroma never develop a cuticular layer
that would impede flow of nutrients and moisture
to the fungus. Through these modifications of the
host tissues, the endophyte removes the barriers
which obstruct nutrient flow into the mycelium.
Very similar to this situation, the cuticle covering
the glandular cells of the Convolvulaceae appears
thinner and therefore more permeable than the
cuticle on epidermal cells.
Clavicipitaceous fungi have evolved to survive
both as saprophytes, degrading organic material,
and as biotrophs of plants, fungi, nematodes and
insects. They are described to have become
particularly successful as endophytes and epi-
bionts of grasses. The associations between clavi-
cipitaceous fungi and their hosts constitute unique
biotrophic symbioses where the stages of physio-
logical adaptation to the plant host may yield an
understanding of how evolution among these
fungi and their hosts has progressed (Schardl
et al. 2008). The detection on Convolvulaceae of
clavicipitaceous fungi able to synthesize ergoline
alkaloids known to play a role in enhanced resis-
tance to diseases, pests and tolerance to drought has
shown that such associations have evolutionary
value not only in grass hosts but also in dicots.
The colonization of a unique plant niche, the cla-
vicipitaceous fungi on Convolvulaceae, represents
a novel finding among beneficial plant–fungus
symbioses in non-graminaceous plants.
2. Phylogenetic Trees
The unusual colony-forming fungus (provisionally
named IasaF13) on the leaf surface of I. asarifolia
(white blooming) was found to belong to the family
Clavicipitaceae. Proof, however, was not possible
by conventional techniques because all attempts to
cultivate the fungus on synthetic media usually
supporting fungal growth were negative. This indi-
cates that the leaf material contains factors or
structures essential for growth of the fungus
IasaF13. All experiments to characterize these
fungi in terms of taxonomy are therefore based
on molecular biological techniques (Steiner et al.
2006).
Construction of phylogenetic trees has been
repeatedly and successfully employed in the sys-
tematic classification of grass-borne clavicipitac-
eous fungi (Spatafora and Blackwell 1993; Glenn
et al. 1996; Kuldau et al. 1997; Reddy et al. 1998;
Lewis et al. 2002; Bischoff and White Jr. 2005;
Sung et al. 2007). Removal of fungal mycelium
from the leaf surface of convolvulaceous plants
was possible by ultrasonic treatment. After DNA
from the fungus was extracted and sequenced
phylogenetic trees were constructed from
18SrDNA and internal transcribed spacer. This
showed that the fungus clustered with clavicipi-
taceous fungi. Confirmation was obtained by par-
tial sequencing with phylogenetic analysis of the
gene (partially) responsible for the committed
step in ergoline alkaloid biosynthesis encoding
the 4-[g,g-dimethylallyl]tryptophan synthase.
Essentially the same results were observed
when the fungi associated with I. asarifolia (red
variety), T. corymbosa, and I. violacea were inves-
tigated (Ahimsa-Mueller et al. 2007). This showed
that our observations are not restricted to one
single plant taxon (I. asarifolia, white blooming)
and its associated fungus IasaF13 but are of a
broader significance at least within the family Cla-
vicipitaceae. The clavicipitaceous fungi present on
representatives of the four convolvulaceous taxa
turned out to be not identical, although closely
related. In-depth investigation of the biosynthetic
pathway leading to ergoline alkaloids in the fungus
present on I. asarifolia (white blooming) provided
further evidence for the clavicipitaceous nature of
the fungus IasaF13 (Markert et al. 2008).
B. Fungicidal Treatment
Discovery of the fungus on the adaxial leaf surface
was a surprise. The hypothesis that an associated
fungus was responsible for the presence of
alkaloids in Convolvulaceae had been tested but
the fungus was not found for unexplained reasons
(Hofmann 2006).
The presence of the clavicipitaceous fungus
alone, however, was not evidence enough to pos-
tulate the fungal origin of ergoline alkaloids in the
symbiotic association. Twelve endophytic fungi
had been isolated from the I. asarifolia Roem. et
Schult plant and an epibiotic fungus – provision-
ally named IasaF13 – was discovered. Not only
was it possible that one of the fungi colonizing
the plant (Steiner et al. 2006) would have been
responsible for the occurrence of alkaloids in
Convolvuaceae plants, it was also possible that
the plant itself was the synthesizing organism.
To clarify this situation I. asarifolia and T.
 Fungal Origin of Ergoline Alkaloids Present in Dicotyledonous Plants (Convolvulaceae)
corymbosa (L.) Raf (syn. Rivea corymbosa (L)
Hall. f.) plants were treated with four different
fungicides in a regime with two-week intervals.
After 18 weeks, microscopic inspection and alka-
loid analysis of plants belonging to both species
revealed that systemic azole fungicides were most
effective in the removal of both epibiotic fungus
and ergoline alkaloids. Simultaneously, the vola-
tile oil present in I. asarifolia was isolated by
steam destillation, and as opposed to ergoline
alkaloids, was found not to be removed during
the fungicide treatment (Kucht et al. 2004). Thus,
the removal of alkaloids is a specific process and
the fungicide does not interfer with the supply of
hemiterpenoid biosynthetic building stones which
are precursors of both ergoline alkaloids (Groeger
and Floss 1998; Keller and Tudzynski 2002) and
terpenoid compounds. This result is a frequent
observation: ergoline alkaloids and the epibiotic
fungus always cooccur because they are both part
of a functional entity (Ahimsa-Mueller et al. 2007).
C. Plant Growth Under Germ-Free Conditions
The notion that fungicides eliminate ergoline
alkaloids from the plant is a clear indication that
ergoline alkaloids in Convolvulaceae plants are of
fungal origin. This observation is somewhat un-
usual because it had been reported that ergoline
alkaloids are produced by plant cell cultures
established from different Convolvulaceae plants
(Dobberstein and Staba 1969). Plant cell cultures
are usually germ-free; they should not contain any
microbes and can therefore be used as a test system
to probe the biosynthetic capacities of plant cells.
Numerous attempts, however, to reproduce
this result (Dobberstein and Staba 1969) and to
find a plant cell culture raised from Ipomoea
asarifolia, Turbina corymbosa and I. violacea
(L) (Convolvulaceae) showing ergoline alkaloid
production were unsuccessful in our hands
(Hussein 2004; Kucht et al. 2004). Indeed, thin-
layer chromatography combined with vanUrk’s
spray reagent were used by Dobberstein and
Staba (1969) to detect ergoline alkaloids, techni-
ques which are of limited reliability in the identi-
fication of natural products (Jenett-Siems et al.
1994; Kucht et al. 2004).
Again, it was a surprise when we found that
the epibiotic fungus lived together with the plant
cells in the callus and cell suspension culture.
203
Microscopic examination, single-strand confor-
mation polymorphism (SSCP) and sequencing of
the internal transcribed spacer revealed the pres-
ence in the cell culture of the epibiotic fungus
(IasaF13) previously detected on the leaf surface
of I. asarifolia. Fungi contaminating plant tissue
cultures usually cause plant cells to react in a
hypersensitive response.
This, however, was not observed in our cultures
where the fungus coexisted asymptomatically and
undetected by the naked eye in association with the
plant cells. Other endophytic fungi which had been
isolated from intact I. asarifolia plants were not
detectable by SSCP within the callus and cell suspen-
sion culture (Steiner et al. 2006).
When a callus culture was subjected to a new
hormone regime (the amount of benzylaminopur-
ine was lowered from 2.0 mg/l to 0.01 mg/l) a
plantlet regenerated from the callus. This plantlet
was colonized by the fungus and contained ergo-
line alkaloids (Steiner et al. 2006, 2008).
These observations show also that an intact
I. asarifolia plant colonized by the fungus IasaF13
is required for the successful synthesis of ergoline
alkaloids and gives an idea about the extreme
specificity in the interaction between the epibiotic
fungus and the I. asarifolia plant (Steiner et al.
2008). It is in line with these conclusions that we
were hitherto unable to grow the fungus IasaF13
in vitro (Steiner et al. 2006). Apparently the plant
contains some kind of component essential for
fungal growth. The specificity between the plant
and its associated fungus is also evident from the
fact that different plant taxa within the Convolvu-
laceae (I. asarifolia, I. violacea, T. corymbosa) are
colonized by related but different clavicipitaceous
fungi (Ahimsa-Mueller et al. 2007).
This raises the question as to how the specific
interaction between the fungus and the host plant
is brought about (Steiner et al. 2008). Interesting-
ly, the fungus apparently has a very high affinity to
the secretory glands on the adaxial leaf surface
(Kucht et al. 2004).This seems to be unusual be-
cause essential oils may have an antifungal activity
(Chang et al. 2008). It is conceivable that during
evolution clavicipitaceous fungi were able to over-
come this barrier and to take advantage of volatile
oil components, using these compounds as med-
iators of specificity and even as substrates to feed
upon.
The volatile oil of I. asarifolia consists of
many minor but five major components, the latter
204 Eckhard Leistner and Ulrike Steiner
of which are sesquiterpenes (Kucht et al. 2004).
Sesquiterpenes play an important role in ecologi-
cal interactions between plants and insects
(Schnee et al. 2006; Gershenzon and Dudareva
2007). Our observations raise the question wheth-
er this class of terpenoids is also essential for the
interaction between different Convolvulaceae spe-
cies and their associated clavicipitaceous fungi.
D. Biosynthesis and Accumulation of Ergoline
Alkaloids in the Fungus/Plant Symbiotum
Ergoline alkaloids are natural products of high
physiological activity. They are likely to confer
drought resistance, herbivore deterrence and fit-
ness to the host plant (Malinowski and Belesky
2000; White Jr. et al. 2003; Bacon and Lyons 2005;
Gershenzon and Dudareva 2007). This raises the
question as to how this may be brought about when
plant-associated clavicipitaceous fungi are the site
of ergoline alkaloid biosynthesis. Indeed, Convol-
vulaceae plants do not seem to have the biosynthet-
ic capacity to produce ergoline alkaloids: neither
the genes nor the enzymic machinery were detect-
able in the shoots. The genetic material responsible
for ergoline alkaloid biosynthesis was clearly found
in the associated fungi present on I. asarifolia
and T. corymbosa (Markert et al. 2008). The
determinant step in ergoline alkaloid biosynthesis
is the prenylation in the 4 position of tryptophan
catalyzed by 4-[g,g-dimethylallyl] tryptophan
synthase (DmaW; Groeger and Floss 1998; Keller
and Tudzinski 2002). The encoding gene – which
has different synonyms, i.e. dmaW or cpd1
(Schardl et al. 2006) or fgaPT2 (Unsöld and
Li 2005) – is clearly present in the fungus and is
part of a cluster in which the ergoline alkaloid
genes are oriented. This is found in Claviceps but
is different from Aspergillus and Neotyphodium
species (Markert et al. 2008). A reverse genetics
experiment showed that the fungus is also the site
of transcription of the dmaW gene (Markert et al.
2008).
Initial attempts to detect ergoline alkloids in
the fungal mycelium present on I. asarifolia and T.
corymbosa failed although two different analytical
approaches were used (Markert et al. 2008). When
a sample of the mycelium found on T. corymbosa
was directly placed into the injection port of a GC/
MS system a trace of agroclavine was detectable
and clearly identified by comparison with an au-
thentic sample. No alkaloid was detectable when a
mycelial sample from I. asarifolia was checked in
the same way (W. Boland, personal communica-
tion). When the leaf material was analyzed for
ergoline alkaloids after removal of the mycelium
by ultrasonic treatment, alkaloids were qualita-
tively and quantitatively detected in the plant
material, showing that the plant leaf material
contained almost all alkaloids whereas the pro-
ducing fungus provisionally named TcorF01 con-
tained only a trace of agroclavine (Markert et al.
2008).
Thus, biosynthesis of alkaloids takes place
in the mycelium; however, ergoline alkaloids
accumulate in the host plant. We have to postulate
a transport system that translocates ergoline alka-
loids from the mycelium into the plant tissue.
In an experimental system similar to the one dis-
cussed here transport was postulated to occur
through the apparently intact cuticle (Smith et al.
1985).
E. Seed Transmittance of Epibiotic Fungi
Colonizing Convolvulaceae
The genus Ipomoea comprises 600–700 pantropi-
cal species. Over half of them are concentrated in
the Americas. The American species are mostly
native but a few have been introduced (Austin and
Huáman 1996). The classification of Ipomoea spe-
cies is still under discussion (Amor-Prats and
Harborne 1993; Austin and Huáman 1996).
There may be multiple reasons for this: Some
species are only endemic and the description of
species is incomplete, especially those native to
Brazil. Many have not been validly described
or are even undiscovered. The genus Ipomoea
consists of three subgenera, i.e. subgenus Erios-
permum, subgenus Ipomoea and subgenus Qua-
moclit. Amor-Prats and Harborne (1993)
attempted to find chemotaxonomic support for
an infrageneric classification of the genus Ipo-
moea by analyzing seeds from 43 species
for their ergoline alkaloid content. The alkaloid-
bearing species fall, however, into each of the
taxonomically defined subgenera.
Hence, there is no clear relationship between the
distribution of alkaloids and the infrageneric classi-
fication of the genus Ipomoea. The whole problem is
clouded by the inability to reproduce published
analytical data in many cases. Consequently, Eich
 Fungal Origin of Ergoline Alkaloids Present in Dicotyledonous Plants (Convolvulaceae)
(2008) lists ergoline positive versus ergoline nega-
tive reports and Amor-Prats and Harborne (1993)
believe that ‘‘the methods of analysis varied leading
to some uncertainty’’.
The reason for the inconsistent picture very
likely depends on the (until recently unknown)
presence of clavicipitaceous fungi on convolvula-
cous plants and within seeds (Kucht et al. 2004;
Steiner et al. 2006; Ahimsa-Mueller et al. 2007)
and the capacity of these fungi to synthesize
ergoline alkaloids (see below).
A freshly harvested and surface-sterilized seed
grown under germ-free conditions gives a plant
colonized by the epibiotic clavicipitaceous fungus.
This plant contains ergoline alkaloids. The epibio-
tic fungus is the only fungus that is detectable by
SSCP on this particular plant. Such a fungus is
detectable in seeds of I. asarifolia and I. violacea
(Steiner et al. 2006; Ahimsa-Mueller et al. 2007).
This shows that the fungus is seed-transmitted
and points to the host specificity typical of asexual
clavicipitaceous fungi (see above).
The viability of the seed-transmitted fungus
very likely is limited and depends on seed age and
storage (Schardl 1994) as well as moisture and
storage temperature (Welty et al. 1987). An
I. violacea plant devoid of ergoline alkaloids and
derived from an alkaloid- and clavicipitaceous
fungus-containing seed was recently described
(Ahimsa-Mueller et al. 2007). In this particular
case the viability of the seed exceeded the viability
of the inhabiting fungus.
It follows that the presence in a convolvulac-
eous plant of ergoline alkaloids may be an unsuit-
able character for taxonomic classifications.
IV. Additional Fungus/Plant Symbiota
in Dicotyledonous Plants
An interesting association consisting of Ipomoea
batatas (L.) Lam. (i.e. sweet potato; Convolvula-
ceae) and Fusarium lateritium Nees: Fr has also
been reported. As described for our clavicipitac-
eous fungi (Sect. III) F. lateritium is primarily
located between the halves of young unfolded
leaves of the I. batatas plant (Hyun and Clark
1998). Yet there is another feature of this fungus/
plant association which we also observed in our
system (Sect. III): The fungus is associated on the
phylloplane with pearl glands and is located
205
around bases of trichomes (Clark 1992). The fun-
gus apparently produces trichothecenes and pro-
tects the host plant against infection by
pathogenic F. oxysporum f. sp. batatas (Wollenw.)
W.C. Snyder & H.N. Hans. However, the asso-
ciated F. lateritium may also be the cause of chlo-
rotic leaf distortion (CLD) disease mediated by
trichothecenes (Clark 1994). After light activation
of trichotecenes during prolonged exposure of the
plant to sunlight CLD occurs. Plants usually re-
cover when cloudy weather prevails. Thus, the
associated fungus may exert a beneficial and a
detrimental effect on the host plant and in both
cases trichothecenes are likely to be the causative
agent.
Two new clavicipitaceous fungi belonging to a
newly established genus (Hyperdermium) were
isolated from an unidentified Asteraceae plant
(genus Bernonia).The fungi were named H. berto-
nii (Speg.) J. White, R. Sullivan, G. Bills et
N. Hywel-Jones and H. pulvinatum J. White, R.
Sullivan, G. Bills et N. Hywel-Jones. As with the
clavicipitaceous fungi described in Sect. III, the
fungi are epibiotic. They belong to the subfamily
Cordycipitoideae (Sullivan et al. 2000).
An entirely superficial mycelium was ob-
served on a South American Asteraceae plant,
Baccharis coridifolia D.C. The endophyte belongs
to the Hypocreales, an order which accommodates
also the family Clavicipitaceae. The fungus occurs
not only epibiotically but also in meristematic
tissue of leaf primordia. No reproductive struc-
tures were detectable. The plant is toxic and it was
assumed that the epibiont is a trichothecene pro-
ducer. Since this fungus and graminaceous Clavi-
cipitaceae (Sect. II) are not closely related,
colonizations (that must have occurred during
evolution) were assumed to be distinct events
(Bertoni et al. 1997).
The same conclusion was drawn for a Mentha
piperita L. plant colonized by a pyrenomycete
which is also associated with glandular trichomes
(Mucciarelli et al. 2002), a striking observation
which led to speculations about the possible func-
tion of the secretory glands and trichomes in the
establishment of a symbiotic association: it may
be that the glandular trichomes are entry gates for
the fungus in its attempt to establish a molecular
dialog with the host plant (Steiner et al. 2008).
Another interesting fungus/plant association
has been described for locoweed plants belonging
to the family Fabaceae. Astragalus molissimus,
206 Eckhard Leistner and Ulrike Steiner
Oxytropis lambertii and O. sericea are collectively
called locoweed and are colonized by endophytes
which seem to be closely related to the genus
Embellisia. Locoism, as observed in cattle intoxi-
cated by locoweed plants, is a neurological disease
resulting in a staggering walk and lack of muscu-
lar coordination. The causative agent seems to be
the indolizidine alkaloid swainsonine. This alka-
loid is also known to be a product of in vitro
grown Rhizoctonia leguminicola cultures. Again
there are observations that parallel those de-
scribed in Sect. III:
1. Some collections of host plants were devoid of
natural products.
2. The fungus and the alkaloid do not occur in the
root system.
3. The alkaloid exerts an ecological function in
protecting the fungus against insects and ani-
mal feeding (Braun et al. 2003).
V. Conclusions
The data described in Sect. III solve a historical
mystery and explain why ergoline alkaloids occur
in disjointed taxa, clavicipitaceous fungi and con-
volvulaceous plants. They dispute the possibility
that during evolution a horizontal transfer of
genes responsible for the synthesis of ergoline alka-
loids occurred from fungi to plants. They also show
that there is no necessity to invoke a repeated
invention of the ergoline alkaloid biosynthetic
pathway during evolution. In fact genes present in
IasaF13, TcorF01, Claviceps purpurea, C. fusiformis,
Balansia obtecta, Neotyphodium coenophialum as
well as Asperillus fumigatus involved in the biosyn-
thesis of ergoline alkaloids share a high similarity
(Markert et al. 2008). It is now evident that clavici-
pitaceous fungi do not only colonize Poaceae and
related monocots but also Convolvulaceae. It is also
clear that the association between fungus and con-
volvulaceous plant is asymptomatic and that a mo-
lecular dialog occurs between associated fungi and
convolvulaceous plants, indicating that both are
members of a symbiotum in which biosynthesis
and accumulation of ergoline alkaloids is spatially
separated and sequestered in different but asso-
ciated organisms. One of the unsolved questions
is whether there are also sexual forms of these
vertically transmitted asexual clvicipitaceous
fungi. It is also unknown how the plant-associated
fungi spread within the plant. Despite repeated
attempts to localize hyphea, spores or propagules
within the host plants, endophytic structures of the
fungi have remained undetected until now.
Some of these observations parallel those
made with fungus/plant associations occurring
in the plant families Asteraceae and Fabaceae
and in the plant species I. batatas, as outlined in
Sect. IV. In each case the associated fungus is the
producer of poisonous natural products (tri-
chothecenes, swainsonine) which may benefit
and protect the host plant.
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10 Secondary Metabolites of Basidiomycetes

ANJA SCHÜFFLER1, TIMM ANKE1

CONTENTS c)
Fomitella fraxinea
(Polyporaceae) . . . . . . . . . . . . . . . . . . . . . 219
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 d) Grifola frondosa
II. Secondary Metabolites and Their Biological (Polyporaceae s. lat.) . . . . . . . . . . . . . . 219
Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 e) Leucopaxillus gentianeus
A. Terpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 (Tricholomataceae) . . . . . . . . . . . . . . . 219
1. Sesquiterpenoids . . . . . . . . . . . . . . . . . . . . . . . 211 f ) Clavariadelphus truncatus
a) Resupinatus leightonii (Clavariaceae) . . . . . . . . . . . . . . . . . . . . . 220
(Tricholomataceae) . . . . . . . . . . . . . . . 211 B. Polyketides, Fatty Acid Derivatives . . . . . . . . 220
b) Conocybe siliginea a) Boreostereum vibrans
(Bolbitiaceae) . . . . . . . . . . . . . . . . . . . . . . 212 (Stereaceae) . . . . . . . . . . . . . . . . . . . . . . . . 220
c) Ripartites tricholoma and b) Junghuhnia nitida
R. metrodii (Paxillaceae) . . . . . . . . . . 212 (Steccherinaceae) . . . . . . . . . . . . . . . . . 221
d) Omphalotus olearius, O. nidiformis c) Ceriporia subvermispora
(Omphalotaceae) . . . . . . . . . . . . . . . . . . 213 (Polyporaceae s. lat.) . . . . . . . . . . . . . . 221
e) Radulomyces confluens d) Suillus luteus (Boletaceae) . . . . . . . . 221
(Corticiaceae s. lat.) . . . . . . . . . . . . . . . 213 e) Hygrophorus spp.
f) Gloeophyllum sp. (Hygrophoraceae) . . . . . . . . . . . . . . . . . 221
(Gloephyllaceae) . . . . . . . . . . . . . . . . . . . 214 f) Trametes menziesii
g) Bovista sp. (Lycoperdaceae) . . . . . . 214 (Polyporaceae s. lat.) . . . . . . . . . . . . . . 221
h) Marasmius sp. g) Gerronema sp.
(Tricholomataceae) . . . . . . . . . . . . . . . 214 (Tricholomataceae) . . . . . . . . . . . . . . . 222
i) Dichomitus squalens h) Imperfect Basidiomycete
(Polyporaceae) . . . . . . . . . . . . . . . . . . . . . 214 Strain 96244 . . . . . . . . . . . . . . . . . . . . . . . 222
j) Macrocystidia cucumis i) Pterula sp. (Pterulaceae),
(Tricholomataceae) . . . . . . . . . . . . . . . . 214 Mycena spp. (Tricholomataceae) . . . 222
k) Creolophus cirrhatus j) Terphenyls from Thelephora
(Hericiaceae) . . . . . . . . . . . . . . . . . . . . . . 216 aurantiotincta, T. terrestris,
l) Coprinus sp. (Coprinaceae) . . . . . . . 216 Hydnellum caeruleum,
m) Dacrymyces sp. Sarcodon leucopus, S. scabrosus
(Dacrymycetaceae) . . . . . . . . . . . . . . . 216 (all Thelephoraceae), and Paxillus
n) Limacella illinita curtisii (Paxillaceae) . . . . . . . . . . . . . . . 222
(Amanitaceae) . . . . . . . . . . . . . . . . . . . . . 217 C. Compounds of Unclear Biogenetic
o) Boletus calopus (Boletaceae) . . . . . 217 Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
p) Russula lepida (Russulaceae) . . . . 217 a) Tremella aurantialba
2. Diterpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 (Tremellaceae) . . . . . . . . . . . . . . . . . . . . . 223
a) Sarcodon scabrosus b) Stereum hirsutum (Stereaceae) . . . . 223
(Thelephoraceae) . . . . . . . . . . . . . . . . . . 217 c) Antrodia serialis (Polyporaceae
b) Mycena tintinnabulum s. lat.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
(Tricholomataceae) . . . . . . . . . . . . . . . 217 d) Aporpium caryae
3. Triterpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 (Tremellaceae) . . . . . . . . . . . . . . . . . . . . 224
a) Irpex sp. (Steccherinaceae) . . . . . . . 218 e) Bondarzewia montana
b) Favolaschia spp. (Bondarzewiaceae) . . . . . . . . . . . . . . . . 224
(Favolaschiaceae) . . . . . . . . . . . . . . . . . 218 f) Cortinarius sp. (Cortinariaceae) . . . 224
g) Chamonixia pachydermis
(Boletaceae) . . . . . . . . . . . . . . . . . . . . . . . . 224
h) Serpula himantoides
1
(Coniophoraceae) . . . . . . . . . . . . . . . . . 225
Institut für Biotechnologie und Wirkstoff-Forschung e.V./Institute i) Pholiota spumosa
for Biotechnology and Drug Research, Erwin-Schroedinger-Strasse (Strophariaceae) . . . . . . . . . . . . . . . . . . . 225
56, 67663 Kaiserslautern, Germany; e-mail: anke@rhrk.uni-kl.de

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
210 Anja Schüffler and Timm Anke

D. Amino Acid Derivatives . . . . . . . . . . . . . . . . . . . . 225 entities among the metabolites of actinomycetes

a) Aporpium caryae and other bacterial sources. This was furthered by
(Tremellaceae) . . . . . . . . . . . . . . . . . . . . . 226 the recent progress in cultivation techniques and
III. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 the development of fast methods for the isolation
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
and structural determination of natural com-
pounds. The following chapter mainly deals with
compounds described from 1998 on and focuses
on biologically active metabolites. Some classes of
I. Introduction fungal metabolites which presently are used as
medical, veterinary, or agricultural antibiotics
Basidiomycetes, a major class of higher fungi were dealt with in the chapter “Non-b-Lactam
adapted to many different climates, habitats, and Antibiotics” of The Mycota, Vol. X (2002). For
substrates have developed a rich and very diverse earlier reviews on bioactive natural products of
secondary metabolism. Its products differ in bio- basidiomycetes and bioactive sesquiterpenes of
genetic origin and structure remarkably from the fungi the reader is referred to Lorenzen and
metabolites of ascomycetes or other prolific pro- Anke (1998), Abraham (2001), and Liu (2005).
ducers of secondary metabolites like actinomy-
cetes or myxobacteria. There are, however, some
similarities to the products of plants, especially
with regard to some polyketides, acetylenes, and II. Secondary Metabolites and Their
sesquiterpenoids. The first systematic investiga- Biological Activities
tions of basidiomycete metabolites originated
after the discovery and introduction into clinical Basidiomycetes like other saprophytic or soil-inha-
practise of penicillin, the first antibiotic not biting microorganisms are prolific producers of
derived by chemical synthesis. Its great success secondary metabolites. Their mycelia and fruiting
initiated a real “gold rush” in the search for new bodies are exposed to a number of predators or
antibiotics and prospective producers. From 1940 competitors, which in some cases can explain the
until the early 1950s mycelial cultures or fruiting production of antibiotics, insecticides, or feeding
bodies of more than 2000 basidiomycetes were deterrents. Among the antibiotics the a-methylene
screened for the production of antibiotics by the lactones and ketones have a very broad and unspe-
pioneering groups of M. Anchel, A. Hervey, F. cific action against prokaryots and eukaryots while
Kavanagh, W. J. Robbins, and W. H. Wilkins (for others are highly selective with regard to their
reviews, see Florey et al. 1949; Wilkins and Harris biochemical targets and organisms. As demon-
1944). Their investigations resulted in the discov- strated for the producers of antifungal strobilurins,
ery of pleuromutilin, the lead compound for the antibiotic production can be stimulated severalfold
semisynthetic tiamulin used in veterinary practise in the presence of other, possibly competing, fungi
and recently also in humans (Kavanagh et al. 1951; (Kettering et al. 2004). In many cases, however, a
Högenauer 1979; Daum et al. 2007). This system- possible benefit for the producing fungus is not
atic search came to an end after Waksman’s dicov- obvious. This is especially true for secondary meta-
ery of the streptomycetes as the most prolific bolites for which up to now no antibiotic or other
producers of antibiotic metabolites. These soil biological activities have been detected. According
bacteria are easily obtained and can be grown in to Zähner et al. (1983) these could be part of a
simple media in large fermenters which greatly still ongoing “evolutionary playground” providing
facilitates the discovery and production process. new chemical solutions for an improved fitness of
Meanwhile the basidiomycetes and mainly their the producing organisms. This contribution covers
fruiting bodies attracted the interest of natural metabolites derived from basidiomycetes and their
products chemists as a source of toxins (Bresinsky biological activities published from 1998 until
and Besl 1985), hallucinogens (Schultes and Hof- 2008. The compounds are arranged by their pre-
mann 1980), and pigments (Gill and Steglich 1987; sumed biogenetic origin and the producing fungi.
Gill 1999). A systematic screening of mycelial cul- Synthetic approaches are included when they have
tures only regained interest when it became in- contributed to elucidate the (absolute) configura-
creasingly difficult to discover new chemical tion or structure–activity studies.
 Secondary Metabolites of Basidiomycetes 211

A. Terpenoids isolated in different screenings because of their

Terpenoids are among the prominent metabolites
of basidiomycetes. Most of these have structures
not encountered elsewhere in nature, with the
notable exception that some sesquiterpenes have
ring structures also found in higher plants, e.g.
caryophyllanes and acoranes (Lorenzen and Anke
1998; Abraham 2001). Geosmin (Fig. 10.1), with its
characteristic musty-earthy odor and first de-
scribed as a typical streptomycete metabolite,
has now been identified as responsible for the
characteristic odor of Cortinarius herculeus (Cor-
tinariaceae), Cystoderma amianthinum (Agarica-
ceae), and Cy. carcharias (Breheret et al. 1999).
1. Sesquiterpenoids
Basidiomycetes are prolific producers of sesqui-
terpenoids. Many of these are readily detected and
OH
OH
OH
unusually high antibiotic and cytotoxic activities.
These are often due to a high chemical reactivity
which, however, makes these compounds less de-
sirable as possible lead structures.
a) Resupinatus leightonii (Tricholomataceae)
1(10),4-Germacradiene-2,6,12-triol, a new germa-
crane sesquiterpene, and 1,6-farnesadiene-3,10,
11-triol, a known nerolidol derivative, were isolated
from submerged cultures of Resupinatus leightonii.
Both compounds (Fig. 10.1) inhibited the cAMP-
induced appressorium formation in Magnaporthe
grisea and showed cytotoxic activity. The forma-
tion of melanized appressoria is a prerequisite for
the invasion of host plants by M. grisea. Inhibitors
are considered to be valuable tools for investigating
the mechanisms and pathways leading to infection
and could contribute to the lead finding process for
novel fungicides. 1(10),4-Germacradiene-2,6,12-
OH
HO

HO
OH
Geosmin 1(10),4-Germacradiene-2,6,12-triol 1,6-Farnesadiene-3,10,11-triol

OH OH
OH
OH
OH

O O

H H H
OH O
Conocenol A Conocenol B Conocenol C

OH
OH O O
O
H
O O

H O H OH
O

Conocenolide A Conocenolide B 5-Demethylovalicin

OH OH
HO

OH OH OH

OH
Riparol A Riparol B Riparol C

Fig. 10.1. Sesquiterpenoids from Resupinatus leightonii, Conocybe siliginea, and riparols A–C from Ripartites spp.
212 Anja Schüffler and Timm Anke

triol and 1,6-farnesadiene-3,10,11-triol are the first
natural compounds interfering with the cAMP-
mediated signal transduction leading to
appressorium formation in M. grisea. Antifungal
or antibacterial activities were not detected, but
moderate cytotoxic activities were described (Eil-
bert et al. 2000).
b) Conocybe siliginea (Bolbitiaceae)
Six new tremulane sesquiterpenes, conocenols A–C
(Fig. 10.1) and D, conocenolide A, and conoceno-
lide B (Fig. 10.1), have been isolated from cultures
of a Chinese collection of Conocybe siliginea (Liu
D-Z et al. 2007). So far no biological activities have
been reported for these compounds. The first tre-
mulanes have been reported from the wood-rotting
Phellinus tremulae by Ayer and Cruz (1993).
c) Ripartites tricholoma and R. metrodii
(Paxillaceae)
In a screening for new metabolites antibiotic and
cytotoxic activities were detected in extracts of
mycelial cultures of Ripartites tricholoma and R.
metrodii. As so far no secondary compounds have
been described from the genus Ripartites a closer
look at the metabolites seemed to be warranted.
Three new compounds, the illudane riparol A, the
illudalane riparol B and the protoilludane riparol
C (Fig. 10.1), together with 13-oxo-9(Z),11(E)-
octadecadienoic acid (a compound of sunflowers),
psathyrellon A, 5-desoxyilludosin (Fig. 10.2), the
illudane compound 8 (Fig. 10.2), and 5-demethy-
lovalicin (Fig. 10.1) were isolated by Weber et al.
(2006). 5-Demethylovalicin was first described by
Ito et al. (1999). Its production by Chrysosporium
luchnowense was reported by Son et al. (2002). It
is a sesquiterpene and inhibits the human methi-
onine aminopeptidase-2 and the growth of human
endothelial cells. 5-Demethylovalicin was the first
ovalicin derivate reported from a basidiomyete.
Psathyrellon A (illudin C) is a constituent of
Psathyrella pseudogracilis (Bastian 1985) and Cli-
tocybe illudens (Omphalotus olearius; Arnone
et al. 1991). 5-Desoxyilludosin and compound
8 were also described from a Bovista sp. (Rasser
et al. 2002). Riparol C exhibits weak antibacterial

OH O OH

HO

OH OH OH
Gloeophyllol A Gloeophyllol B Gloeophyllol C

HO HO
OH

HO HO
OH
OH O
Gloeophyllol D Gloeophyllone 1-Hydroxy-3-sterpurene

O
OH OH

OH O

OH O OH HO OH O
O O
Bovistol Psathyrellon B Dimer of Psathyrellon B

OH OH

OH R OH

OH O
HO
Fig. 10.2. Sesquiterpenoids 5-Desoxyilludosin, R=H
from Omphalotus olearius, O. 13-Hydroxy-5-desoxyilludosin, R=OH
nidiformis, Mycena leaiana,
OH
Radulomyces confluens, and the
synthetic (
)-irofulven Drimene-2,11-diol Compound 8
 Secondary Metabolites of Basidiomycetes 213

and antifungal activities while riparol A inhibits
the growth of MDA-MB-231 (human breast ade-
nocarcinoma) and MCF-7 (human breast adeno-
carcinoma) cells at concentrations of 1 mg/ml.
d) Omphalotus olearius, O. nidiformis
(Omphalotaceae)
Omphalotus olearius (syn. Clitocybe illudens, O.
illudens) is a rich source of sesquiterpenoids with
carbon skeletons named after the original producer
(e.g. illudane, protoilludane, illudalane). To these a
new sesquiterpenoid, omphadiol (Fig. 10.3), with a
carbon skeleton and also found among the meta-
bolites of the soft coral Lemnalia africana and the
ascomycete Leptographium lundbergii was added
recently (McMorris et al. 2000). It is highly remark-
able that a synthetic analog of illudin S, (
)-iroful-
ven (Fig. 10.3; Mc Morris et al. 2004), has entered
phase II clinical trials and demonstrated activity in
ovarian, gastrointestinal, and non-small cell lung
cancer. As compared to the natural product, iro-
fulven has a much better therapeutic index and
pharmacological profile (Paci et al. 2006). Leaiana-
fulven (Fig. 10.3), a natural product of Mycena
leaiana (Harttig et al. 1990), shares high cytotoxic
activities and a preferential inhibition of nucleic
acid synthesis with irofulven. O. nidiformis is
native to Australia and usually found in eucalypt
forests. From submerged cultures of this species
three new iludins (illudin F, G, H; Fig. 10.3) so far
not found in the North American species have been
isolated (Burgess 1999).
e) Radulomyces confluens (Corticiaceae s. lat.)
Radulone A, radulone B, and radudiol, the illuda-
lane radulactone and the illudane radulol

HO
O O
OH HO
OH HO
H H

OH
OH
Omphadiol (-)-Irofulven Leaianafulven

OH O OH O OH O
OH OH OH

HO HO HO
OH

Illudin F Illudin G Illudin H

HO O HO OH

O O OH
Radulone A Radulone B Radudiol

OH

HO O
Radulactone Radulol

Fig. 10.3. Sesquiterpenes from Gloeophyllum sp., Bovista sp., and 5-desoxyilludosin and compound 8 from Riparti-
tes spp.
214 Anja Schüffler and Timm Anke
(Fig. 10.3) were isolated from fermentations of
Radulomyces confluens (Fabian et al. 1998). Radu-
lone A is a potent inhibitor of human and bovine
platelet aggregation stimulated by different ago-
nists, inhibiting preferentially the aggregation of
human platelets induced by ADP with an IC50
value of 2 mM. In addition, radulone A exhibits
cytotoxic and antimicrobial activities. The other
four compounds show weak antimicrobial and
cytotoxic activities.
f) Gloeophyllum sp. (Gloephyllaceae)
Six new sesquiterpenoids, four rearranged illuda-
lanes, one rearranged protoilludane, and one ster-
purane (Fig. 10.2), were isolated from fermentations
of a Gloeophyllum species (Rasser et al. 2000). The
strain was isolated from fruiting bodies collected
from a mangrove in Florida, United States. The
sporophores showed the characteristics of the
genus (Jülich 1984). The morphological character-
istics of the sporophores closely resembled G.
sepiarium for which, however, an occurrence on
mangrove wood was considered unlikely. Gloeo-
phyllol A, gloeophyllol D, and gloeophyllone did
not exhibit antibacterial or antifungal activity.
Gloeophyllols B and C showed weak antifungal ac-
tivity, while 1-hydroxy-3-sterpurene showed weak
antifungal, antibacterial, and cytotoxic activities.
g) Bovista sp. (Lycoperdaceae)
Mycelial cultures of Bovista sp. 96042 were
derived from tissue plugs of a young fruiting
body. The saprophytic soil inhabiting species
showed all characteristics of the genus; the species
however could not be identified. The strain
yielded several cytotoxic sesquiterpenoids (Rasser
et al. 2002). Besides the known illudane psathyr-
ellon B (illudin C3) and protoilludane armillol, 5-
desoxyilludosin and 13-hydroxy-5-desoxyilludo-
sin, two new secoprotoilludanes, the illudane
compound 8 and drimene-2,11-diol, the novel
hexacyclic metabolite bovistol were obtained
(Fig. 10.2). This can formally be regarded as a
triterpene, but appears to be formed from
psathyrellon B by a heteroatom Diels–Alder di-
merization. Indeed, the spontaneous dimerization
of psathyrellon B in methanol resulted in the
formation of the expected dimer (Fig. 10.2).
h) Marasmius sp. (Tricholomataceae)
6,9-Dihydroxy-3(15)-caryophyllen-4,8-dione
(Fig. 10.4), a new caryophyllane sesquiterpene,
was isolated from fermentations of a tropical Mar-
asmius species. Caryophyllane sesquiterpenoids
have been described as widespread constituents
of several plants, e.g. Jasminum, Lavendula, and
Juniperus, and basidiomycetes. 6,9-Dihydroxy-3
(15)-caryophyllen-4,8-dione showed strong cyto-
toxic activities on L1210 (mouse lymphocytic
leukemia) and HL-60 (human promyelocytic leu-
kemia) cells with IC50 values of 1.9 mM and 3.8 mM
but no antimicrobial activity. The aggregation of
human thrombocytes stimulated with ADP or col-
lagen was inhibited with IC50 values of 47 mM
while no inhibition of thrombin induced aggrega-
tion was observed (Fabian et al. 1999).
i) Dichomitus squalens (Polyporaceae)
From mycelial cultures of a Chinese collection of
Dichomitus squalens three new sesquiterpenes
dichomitol (dichomitin A), 2b,13-dihydroxyledol
(dichomitin B), and dichomitone (dichomitin C)
were described by Huang et al. (2004). The struc-
ture of dichomitol is interesting because C-15 is
attached to C-6 instead of C-4 as in other hirsutane
sesquiterpenes. Dichomitol is the first example of
1,10-seco-2,3-seco-aromandendrane sesquiter-
penes. The nomenclature of the compounds is
confusing because in the same paper the three
compounds were given two different names each
(above in brackets; Fig. 10.4). Of the three com-
pounds 2b,13-dihydroxyledol exhibited nematici-
dal activity against Bursaphelenchus xylophilus,
a Pinus pathogen with a LC50 of 35.6 mg/ml.
j) Macrocystidia cucumis (Tricholomataceae)
Eight new triquinane-type sesquiterpenoids were
isolated from fermentations of Macrocystidia cucu-
mis (Hellwig et al. 1998). Cucumins A–D are highly
unsaturated hirsutanes while cucumins E–G pos-
sess a novel carbon skeleton for which the name
cucumane is proposed. Cucumin H is a new cera-
topicane (Fig. 10.4). Three further metabolites
were identified as cyclo(phenylalanylprolyl), cyclo
(leucylprolyl), and arthrosporone. Cucumin A exhi-
bits high antibiotic activity against Bacillus subtilis,
Nematospora coryli, and Mucor miehei at minimal
inhibitory concentrations (MIC) of 1–10 mg/ml,
while cucumin C preferentially inhibits the growth
of N. coryli and Saccharomyces cerevisiae. The anti-
bacterial and antifungal activities of cucumin B are
very weak. However, all three compounds are high-
ly cytotoxic with IC100 = 0.5–1.0 mg/ml. As is the
case with other sesquiterpenes the biological
 Secondary Metabolites of Basidiomycetes 215

HO O H OH
H H
OH
HO H H
HO
H HO H
O H OH OH
6,9-Dihydroxy-3(15)- Dichomitol 2ß,13-Dihydroxyledol
caryophyllen-4,8-dione

O
OH O O

O H O O
H H
H
H OH Cucumin A Cucumin B

Dichomitone
O
O O H

O O O
O H
H OH
Cucumin C Cucumin D Cucumin E

OH O O
H H

O HO
H H
HO
Cucumin F Cucumin G Cucumin H

O H O H O H
OH
O

HO HO HO
O
HO H H
O HO O

Creolophin A Creolophin B Creolophin C

OH
H
O H O H
O
O O O
O
HO O O
H HO
O O O
O OH
Creolophin D Creolophin E Neocreolophin
O
H

Fig. 10.4. Sesquiterpenes from Marasmius sp., Dichomitus squalens, Macrocystidia cucumis and Creolophus cirrhatus
216 Anja Schüffler and Timm Anke

activities are closely correlated to their reactivity l) Coprinus sp. (Coprinaceae)

towards nucleophiles like cysteine.
k) Creolophus cirrhatus (Hericiaceae)
Five new norhirsutanes with 14 carbon atoms,
instead of 15 as in the hirsutanes, were isolated
from fermentations of Creolophus cirrhatus iso-
lated from a fruiting body collected in France
(Opatz et al. 2007; Birnbacher et al. 2008). Creolo-
phins A–E (Fig. 10.4) were isolated together with
the known hirsutane complicatic acid. Only creo-
lophin E and its dimer neocreolophin (Fig. 10.4B)
which forms as an artefact during purification
showed antibiotic and cytotoxic activity. In con-
trast to creolophins A–D, which exhibit no antibi-
otic and cytotoxic activities, creolophin E and its
dimer possess a highly reactive exomethylene ke-
tone moiety which readily reacts with nucleo-
philes like cysteine, resulting in a complete loss
of activity (Birnbacher et al. 2008).
Coprinol, a new cuparane (Fig. 10.5), was isolated
from fermentations of a wood inhabiting Coprinus
sp. (Johansson et al. 2001). The new antibiotic
exhibited activity against Gram-positive multiresis-
tant strains, including penicillin-resistant pneumo-
cocci (PRSP), methicillin- and quinolone-resistant
staphylococci (MRSA, QRSA), vancomycin-
resistant enterococci (VREF), and vancomycin
intermediate-resistant staphylococci (VISA). Copri-
nol exhibited no activity against Gram-negative
bacteria and fungi.
m) Dacrymyces sp. (Dacrymycetaceae)
Two new antibiotic metabolites, the eremophilane
dacrymenone and VM 3298-2 (Fig. 10.5) were
isolated from fermentations of a Dacrymyces sp.
collected in La Réunion, France (Mierau et al.
2003). Dacrymenone inhibits the growth of bacte-
ria with MIC values of 25-100 mg/ml (86–343 mM).

O O
HO OH
HO O
O
O
HO
O
O
O
Dacrymenone
OH OH
Coprinol VM 3298-2

O HO OH
O O

O O
O HO

O
PR-Toxin H Petasol 11-Desoxyeleganthol

O O
O OH O
O OH
O
O HO HO
HO HO
Limacellone Illitinone A Illitinone B
O O
O H O H NH2 O
O O O O
OH OH
Fig. 10.5. PR toxin, petasol, and H
OAc
H
OH
sesquiterpenes from Coprinus
sp., Dacrymyces sp., Limacella O O
illinata, Boletus calopus, and
Russula lepida O-Acetylcyclocalopin A Cyclocalopin A Lepidamine
 Secondary Metabolites of Basidiomycetes
VM 3298-2 exhibits weak antifungal activity
against yeasts and filamentous fungi, with the
highest activity on Candida parapsilosis at 25 mg/
ml (110 mM) and S. cerevisiae at 10 mg/ml (44 mM).
PR toxin (Fig. 10.5) from Penicillium roquefortii is
described to have strong cytotoxic, fungicidal, and
bactericidal properties (Moulé et al. 1977). The
greatly reduced antibiotic activities and the lack
of cytotoxicity of dacrymenone are likely due to
the lack of the aldehyde group and the oxirane
rings present in PR toxin. VM 3298-2 is cytotoxic
towards Colo-320 (human colon adenocarcino-
ma) with IC50 values of 10 mg/ml (44 mM) and 5
mg/ml (22 mM) for HL-60 and L1210 cells. Related
eremophilans, e.g. petasol (Fig. 10.5), produced by
fungi and plants have phytotoxic effects (Suga-
wara et al. 1993). When tested in an assay using
leaf cuts of Hordeum sativum, concentrations as
low as 1 mM dacrymenone produced green islands
(20 mm2) and necrotic lesions on leafs, whereas in
a similar assay petasol was reported to produce
green islands on monocot leafs at much higher
concentrations (10–20 mM; Sugawara et al. 1993).
n) Limacella illinita (Amanitaceae)
In a search for new bioactive compounds from basi-
diomycetes, four new compounds (Fig. 10.5) were
isolated from fermentations of Limacella illinita
(Gruhn et al. 2007). Limacellone has a new C15
carbon skeleton. Limacellone and illinitones A and
B share large parts of the carbon skeleton and are
therefore considered to be biogenetically related.
11-Desoxyeleganthol belongs to a group of sesqui-
terpenes which have been described from higher
plants, but are quite rarely isolated from fungi. It is
similar to eleganthol from Clitocybe elegans
(Arnone et al. 1993). Illinitone A exhibits weak phy-
totoxic and moderate nematicidal activities against
Caenorhabditis elegans, illinitone B is moderately
cytotoxic, while limacellone exhibits weak cytotoxic
and phytotoxic activities. 11-Desoxyeleganthol was
found to be inactive in all assays. No biological
activities have been described for eleganthol so far,
which is in accordance with our findings.
o) Boletus calopus (Boletaceae)
Bitter tasting boletes are a nuisance to all mush-
room hunters and gourmets who inadvertently
add a fruiting body of, e.g. Boletus calopus, to a
meal of B. erythropus or B. edulis. In fruiting
bodies of B. calopus and closely related mush-
rooms (B. radicans, B. coniferarum, B. rubripes,
217
B. peckii) O-acetylcyclocalopin A (Fig. 10.5) was
identified as the main bitter principal by Hellwig
et al. (2002). It was accompanied by the slightly
bitter cyclocalopin A and a number of other meta-
bolites. Interestingly, no calopins could be
detected in extracts of the bitter bolete Tylopilus
felleus, a look-alike of B. edulis.
p) Russula lepida (Russulaceae)
A number of new terpenoids have been isolated
from fruiting bodies of Russula lepida. Among
them are lepidamine (Fig. 10.5), the first natural
aristolane-type sesquiterpene alkaloid (Tan et al.
2003), and among other triterpenoids lepidolide
(Tan et al. 2002) a cucurbitane (Fig. 10.6). No data
on biological activity are available.
2. Diterpenoids
Diterpenoids are less frequently encountered
among the basidiomycete metabolites. However
some of them have interesting ring systems so
far not found elsewhere. A semisynthetic pleuro-
mutilin has even become a product used in veteri-
nary medicine.
a) Sarcodon scabrosus (Thelephoraceae)
The cyathins and striatins are prominent products
of Cyathus spp. Some of them have very high cyto-
toxic activities. Interesting recent additions to this
class of compounds are the sarcodonins (Shibata
et al. 1998) and the scabronines (Kita et al. 1998;
Ohta et al. 1998; Ma et al. 2004) from Sarcodon
scabrosus. The sarcodonins (e.g. sarcodonin A;
Fig. 10.6) have a bitter taste, antiinflammatory and
antibacterial activities (Shibata et al. 1998; Hirota
et al. 2002; Kamo et al. 2004). Interesting pharma-
cological activities have been reported for scabro-
nines. Scabronines A and G (Fig. 10.6) have been
shown to promote the secretion of neurotrophic
factors, including nerve growth factor from
1321N1 (human astrocytoma) cells, causing the en-
hancement of differentiation (neurite outgrowth) of
PC-12 (pheochromocytoma of rat adrenal medulla)
cells (Obara et al. 1999). Obara et al. (2001) sug-
gested that scabronine G and its methylester would
enhance the secretion of neurotrophic factors from
1321N1 cells by activation of protein kinase C-z.
b) Mycena tintinnabulum (Tricholomataceae)
Fruiting bodies and mycelia of Mycena tintinnabu-
lum grown in complex media or on oak wood
218 Anja Schüffler and Timm Anke

O OH O O
Fig. 10.6. Diterpenoids and tri-
terpenoids from Sarcodon scab-
rosus, Mycena tintinnabulum, OH H O H O
Russula lepida, and Irpex sp.

HO O O O
Sarcodonin A Scabronine G Scabronine G methylester

O
OH

OH O H OH

HO

O
O
Tintinnadiol O Lepidolide

OH
HO
OH
O
HO
14-Acetoxy-15-dihydroxyirpexan
HO O

O OH

O
OH
HO
OH
O
HO
14,15-Dihydroxyirpexan
HO O

OH OH

contained antifungal strobilurins, whereas tintin-
nadiol (Fig. 10.6), a new sphaeroane diterpene, was
isolated only from the fruiting bodies. Tintinnadiol
exhibited cytotoxic activities towards HL-60 cells
(IC50 = 10 mg/ml) and L1210 cells (IC50 = 40 mg/ml).
In the agar diffusion assay, the compound showed
neither antifungal nor antibacterial activity at con-
centrations up to 50 mg/disc (Engler et al. 1998).
3. Triterpenoids
Basidiomycetes are prolific producers of triterpe-
noids, among them many with interesting biological
activities.
signal transduction pathways (Silberborth et al.
2000). Both pathways play major roles in inflamma-
tion and cancer. The irpexans consist of a triterpe-
noid chain with an attached mannose moiety.
14-acetoxy-15-dihydroxyirpexan (Fig. 10.6) inhib-
ited the phorbol ester induced expression of an
AP-1 or NF-kB dependent reporter gene with
IC50 values of 6–7 mg/ml followed by 14,15-irpexan-
oxide, 14,15-dihydroxyirpexan (Fig. 10.6) and
14-acetoxy-22,23-dihydro-15,23-dihydroxyirpexan,
whereas irpexan exhibited no activity. All isolated
compounds showed no antibacterial or antifungal
activity. Cytotoxic effects were observed starting at
20 mg/ml.

a) Irpex sp. (Steccherinaceae) b) Favolaschia spp. (Favolaschiaceae)

The irpexans were isolated in the course of a screen- The genus Favolaschia is a rich source of biolog-
ing for new inhibitors of AP-1 and NF-kB mediated ically active compounds. Among them are
 Secondary Metabolites of Basidiomycetes
strongly antifungal strobilurins and oudeman-
sins (Zapf et al. 1995; Wood et al. 1996; Nicholas
et al. 1997), (+)-10a-hydroxy-4-muurolen-3-one,
an inhibitor of leukotriene biosynthesis (Zapf
et al. 1996), favolon A (Anke et al. 1995), and
laschiatrion (Anke et al. 2004). Laschiatrion
(Fig. 10.7), a new antifungal antibiotic, was
isolated from fermentations of Favolaschia sp.
87129. It possesses a new steroid skeleton. Laschia-
trion exhibits broad in vitro antifungal activity
against Candida albicans, Cryptococcus neofor-
mans, Aspergillus flavus, Fusarium verticillioides,
Trichophyton mentagrophytes, and Microsporum
gypseum at concentrations of 10–50 mg/ml. No
antibacterial and cytotoxic activities could be
detected. By comparison, favolon A, an antifungal
triterpenoid with a different ring system and
substitution pattern exhibits higher activities at
concentrations starting from 1 mg/ml. Recently,
favolon B (Fig. 10.7) was isolated from a Chilean
Mycena species (Aqueveque et al. 2005). It differs
from favolon A in the replacement of favolon’s
epoxide group in the B ring by a double bond. It
shares the high antifungal activities of the parent
compound.
O
O O
HO
O HO
O
OH
HO
Laschiatrion
O O O O
HO
OH OH
H Fomitellic acid A H Fomitellic acid B
O O
HO H OH HO H OH
O O
O
OH OH
H Fomitellic acid C HO
O
H H OH
HO H OH
O O
219
c) Fomitella fraxinea (Polyporaceae)
3b-Hydroxylanosta-8,24-dien-21-oic acid and fomi-
tellic acids A–D (Fig. 10.7 7), four new lanostanes,
were isolated from the mycelia of Fomitella fraxinea
in a search for new inhibitors of DNA polymerases
(Tanaka et al. 1998). All five metabolites inhibited
calf DNA polymerase a and rat DNA polymerase b
at concentrations of 35–75 mM and 90–130 mM
respectively. Data on other biological activities are
not given.
d) Grifola frondosa (Polyporaceae s. lat.)
Many different biological activities have been
ascribed to the common fungal metabolite ergos-
terol, its oxidation products as well as some deri-
vatives. New to the list are inhibitory activities of
ergosterol and ergostra-4,6,8(14),22-tetraen-3-one
(Fig. 10.8) on cyclooxygenase-1 and -2 at rather
high concentrations (250 mg/ml). Both com-
pounds were isolated from cultured mycelia of
Grifola frondosa by Zhang et al. (2002).
e) Leucopaxillus gentianeus (Tricholomataceae)
The cucurbitanes leucopaxillones A and B (Fig.
10.8) have been isolated from fruiting bodies of
HH
Favolon B
O
Fomitellic acid D
O Fig. 10.7. Triterpenoids from
Favolaschia sp. and Fomitella
fraxinea
220 Anja Schüffler and Timm Anke

Fig. 10.8. Triterpenoids from

Grifola frondosa, Leucopaxillus O O
O O
gentianeus, Clavariadelphus
truncatus, and vibralactones HO
OH
from Boreostereum vibrans
H H

H
O O
Ergostra-4,6,8(14),22-tetraen-3-one Leucopaxillone A

HO
O O O
O O
OH
OH
O OH O
HO
OH
H H O

O
O H

Clavaric acid
Leucopaxillone B

H
O O H
O O
R
O O OH
O
H H
Vibralactone R = CH2OH Vibralactone B 1,5-Secovibralactone
Acetyl-vibralactone R = CH2OAc
Vibralactone C R = CHO

Leucopaxillus gentianeus, together with cucurbi- B. Polyketides, Fatty Acid Derivatives

tacin B, a known plant metabolite (Clericuzio et al.
2004). When tested for cytotoxic activities to-
wards the cell lines A549 (human lung carcino-
ma), CAKI 1 (human kidney carcinoma), Hep-G2
(human liver carcinoma), and MCF-7 both leuco-
paxillones compared unfavorably with the known
cytotoxic activities of cucurbitacin B.
f) Clavariadelphus truncatus (Clavariaceae)
Lingham et al. (1998) identified clavaric acid
(24,25-dihydroxy-2-(3-hydroxy-3-methylglutaryl)
anostan-3-one; Fig. 10.8) as a novel metabolite
of Clavariadelphus truncatus. The compound
exhibits very interesting activities. It inhibits
selectively human farnesyl-protein transferase
(FPTase) with an IC50 value of 1.3 mM without
interfering with geranylgeranyl-protein transfer-
ase-I (GGPTase-I) or squalene synthase activity.
It is competitive with respect to Ras and is a
reversible inhibitor of FPTase.
a) Boreostereum vibrans (Stereaceae)
The vibralactones (Fig. 10.8), unusual fused b-
lactone-type metabolites were isolated from cul-
tures of Boreostereum vibrans (syn. Stereum
vibrans; Liu et al. 2006; Jiang et al. 2008).Vibra-
lactone is a potent inhibitor of pancreatic lipase
with an IC50 value of 0.4 mg/ml. A structurally
closely related metabolite, percyquinnin, had pre-
viously been isolated from fermentations of Ster-
eum complicatum as a potent lipase-inhibitor
(Hopmann et al. 2001). It later turned out to be
identical with vibralactone. Four new related
metabolites, 1,5-secovibralactone, vibralactone B,
vibralactone C, and acetylated vibralactone have
now been described from the same fungus. Inhi-
bitors of pancreatic lipase like orlistat, a derivative
of the streptomycete metabolite lipstatin (Weibel
et al. 1987) are now medications for obese patients
(Bray and Greenway 2007). Its mode of action was
 Secondary Metabolites of Basidiomycetes
elucidated by Hadvary et al. (1991). Tetrahydro-
lipstatin binds covalently to the putative active
site serine by means of the b-lactone moiety. The
inhibitory activity of vibralactone may be due to
the same mechanism.
b) Junghuhnia nitida (Steccherinaceae)
Many polyacetylenes have been described from
basidiomycetes. Nitidon (Fig. 10.9), a highly oxi-
dized pyranone derivative produced by Junghuh-
nia nitida, exhibits antibiotic and cytotoxic
activities and induces morphological and physio-
logical differentiation of tumor cells at nanomolar
concentrations (Gehrt et al. 1998). At a concentra-
tion of 100 ng/ml (0.46 mM) nitidon induces the
differentation of 25–30% of the HL-60 cells into
granulocyte-monocyte-like cells and a differenta-
tion of 20% of the U-937 cells (human histiocytic
leukemia) into monocyte-like cells. The biological
activity of nitidon is at least in part due to its high
chemical reactivity. Addition of cysteine yielded
adducts which were almost devoid of differentia-
tion inducing activity.
c) Ceriporia subvermispora (Polyporaceae s. lat.)
Ceriporia subvermispora is a white rot fungus
which degrades lignin without substantially dam-
aging the remaining cellulose. This bears implica-
tions, e.g. for the cellulose and paper industry. The
reason for this selective degadation of lignin was
investigated by Watanabe’s group (Ohashi et al.
2007). They found that alkylitaconic acids, e.g.
ceriporic acid B (Fig. 10.9), suppress the produc-
tion of hydroxyl radicals by the Fenton reaction
(Rahmawati et al. 2005) even in the presence
of reductants for Fe3+. The alkyl side-chain and
the two carboxyl groups are essential for redox
HO
O
HO O
Ceriporic acid B
O
O O Nitidon
OH
Suillumide
NH
O ghuhnia nitida, Ceriporia sub-
OH
221
silencing and high stability against oxidative deg-
radation by OH radicals.
d) Suillus luteus (Boletaceae)
Suillumide, (2R)-N-[(3S*,4S*,5R*)-5-dodecyl-4-
hydroxytetrahydrofuran-3-yl]-2-hydroxyheptade-
canamide, (Fig. 10.9), was isolated from fruiting
bodies of Suillus luteus collected in Colombia
(Léon et al. 2008). So far, ceramides with a tetrahy-
drofuranyl ring have been found only in fungi. Suil-
lumide inhibits the growth of SK-MEL-1 (human
melanoma) cells with an IC50 value of 10 mM.
e) Hygrophorus spp. (Hygrophoraceae)
The hygrophorones (e.g. hygrophorones D12, F12,
A12; Fig. 10.10), new acylpentenones, were isolated
from fruiting bodies of Hygrophorus latitabundus,
H. olivaceoalbus, H. persoonii, and H. pustulatus
(Lübken 2006; Lübken et al. 2004, 2006). All hygro-
phorones exhibited modest antifungal activity
against Cladosporium cucumerinum.
Chrysotriones A and B (Fig. 10.10) were
isolated from the fruiting bodies of the H. chryso-
don (Gilardoni et al. 2007). Both compounds are
new 2-acylcyclopentene-1,3-dione derivatives. The
chrysotriones exhibited modest antifungal activity
against the phytopathogen Fusarium verticillioides.
f) Trametes menziesii (Polyporaceae s. lat.)
The trametenamides A and B (Fig. 10.11), two new
ceramides were isolated from fruiting bodies of
Trametes menziesii collected in Columbia. The
absolute stereochemistry of trametamide B was
determined by total synthesis. Acetyl-trameta-
mide A was cytotoxic towards SK-MEL-1 cells
with an IC50 value of 8 mM by induction of
apoptosis measured by DNA fragmentation,
poly-(ADP-ribose) polymerase cleavage, and
O
OH
Fig. 10.9. Nitidon, ceriporic acid
B, and suillumide from Jun-
vermispora, and Suillus luteus
222 Anja Schüffler and Timm Anke

O
Fig. 10.10. Hygrophorones and O
chrysotriones from Hygrophorus
spp. OH
Hygrophorone D12 O OH
OH
O

O Chrysotrione A
O

HO
Hygrophorone F12 O OH

O Chrysotrione B
O
O O

OH
4,6-Di-O-acetyl hygrophorone A12
O O

procaspase-9 and -8 processing (Léon et al. 2006).
Ceramides are thought to play a role in the induc-
tion of apoptosis by permeabilization of the mito-
chondrial membrane, allowing the exit of
proapoptotic factors like cytochrome c.
g) Gerronema sp. (Tricholomataceae)
The gerronemins (Fig. 10.11) were isolated from
fermentations of a Gerronema species collected in
the United States (Silberborth et al. 2002). They
are composed of a C12–C16 alkane or alkene sub-
stituted at both ends by 2,3-dihydroxyphenyl
groups. The gerronemins blocked the inducible
expression of a human COX-2 and iNOS promoter
driven reporter gene with IC50 values of 1–5 mg/
ml. In addition, cytotoxic activities were observed
which were due the inhibition of cellular macro-
molecular syntheses.
h) Imperfect Basidiomycete Strain 96244
5-(20 -Oxoheptadecyl)-resorcinol and 5-(20 -oxono-
nadecyl)-resorcinol (Fig. 10.12) were isolated from
fermentations of an imperfect basidiomycete (Filip
et al. 2002). This was obtained from pieces of a
pinkish mycelium covering a piece of rotting wood
in Provence, France. The mycelia bore clamp con-
nections but neither basidia nor conidia could be
found. Both resorcinols exhibit cytotoxic effects
against the human colon tumor cell lines Colo-320,
DLD-1 and HT-29 and the human promyeloid leu-
kemia cell line HL-60, the human leukemia T cell
JURKAT, the human hepatocellular carcinoma cell
line Hep-G2 as well as the J774 mouse macrophage
cell line. The compounds induce morphological and
physiological differentiation of HL-60 cells into
granulocytes, which subsequently die by apoptosis.
Both compounds show no antibacterial or antifun-
gal activity. Interestingly 5-(20 -oxoheptadecyl)-res-
orcinol was reported from rye grains (Kozubek and
Tyman 1995) and both compounds are suggested to
be produced by rice (Suzuki et al. 1996).
i) Pterula sp. (Pterulaceae), Mycena spp.
(Tricholomataceae)
A new antifungal (E)-b-methoxyacrylate, norou-
demansin A (Fig. 10.12), was isolated from cul-
tures of Pterula sp. 82168. As compared to
oudemansin A, its antifungal activities are much
lower (Engler-Lohr et al. 1999). Strobilurin N
(Fig. 10.12) was isolated from fruiting bodies of
Mycena crocata (Buchanan et al. 1999). Interest-
ingly, strobilurin N is the first strobilurin without
antifungal activity. The new antifungal strobilur-
ins I from Agaricus sp. 89139 and K from M.
tintinnabulum (Fig. 10.12) are 3,4-dihydro-2H-
benzo[b](1,4)dioxepin derivatives (Hellwig et al.
1999). Both compounds strongly inhibit yeasts
and filamentous fungi.
j) Terphenyls from Thelephora aurantiotincta,
T. terrestris, Hydnellum caeruleum, Sarcodon
leucopus, S. scabrosus (all Thelephoraceae), and
Paxillus curtisii (Paxillaceae)
Terphenyls are derived from the shikimate–chor-
ismate pathway and are among the frequently
 Secondary Metabolites of Basidiomycetes 223

R O Fig. 10.11. Trametamides and

Trametamide A R = H, R1 = H
gerronemins from Trametes men-
C17H35 NH OR1 ziesii and Gerronema sp.
Acetyltrametamide A R = H, R1 = Ac
OH C17H35 Trametamide B R = OH, R1 = H

OR1 OR1

OH
Gerronemin A OH
OH
OH

OH
OH

Gerronemin B
OH
OH

OH
Gerronemin C OH
OH
OH

OH
OH

Gerronemin D
OH
OH

OH
OH
OH
OH Gerronemin E

OH
OH
OH
OH Gerronemin F

encountered basidiomycete pigments (Gill and
Steglich 1987; Gill 1999; Liu 2006). More recent
additions are the thelephantins (e.g. A and H;
Fig. 10.13) from Thelephora aurantiotincta and
Hydnellum caeruleum (Quang et al. 2003a, b,
2004), terrestins from T. terrestris (Radulović
et al. 2005), and curtisians (e.g. A and C;
Fig. 10.13) from Paxillus curtisii which has inhibi-
tory activity against lipid peroxidation with IC50
values of 0.15, 0.17, 0.24, and 0.14 mg/ml (Yun
et al. 2000). A number of biological activities
have been ascribed to members of this group. The-
lephoric acid (Fig. 10.13), the characteristic pig-
ment of many Thelephoraceae, is claimed to be an
antitumor (Kawai et al. 2005) and antiallergic agent
(Tateishi et al. 2005). Sarcodonin (Fig. 10.13), a
very interesting nitrogen-containing terphenyl
derivative with a modified diketopiperazine at-
tached to it was isolated from fruiting bodies of
Sarcodon leucopus (Geraci et al. 2000). S. scabrosus
collected in China yielded sarcodonin d (Fig. 10.13;
Ma and Liu 2005).
C. Compounds of Unclear Biogenetic Origin
a) Tremella aurantialba (Tremellaceae)
Tremellin (Fig. 10.14), a simple, highly symmetric
structure was isolated from fruiting bodies of Tre-
mella aurantialba, an edible mushrooms (Ding
224 Anja Schüffler and Timm Anke

OH

OH

5-(2'-Oxoheptadecyl)-resorcinol OH

OH
5-(2´-Oxononadecyl)-resorcinol

O O
OH
HO OH
O

O O O
O

Noroudemansin A Strobilurin N O

O O

HO O
O O O O
O O
O O
Strobilurin I Strobilurin K

Fig. 10.12. Resorcinols, noroudemansin A and strobilurins from an imperfect basidiomycete, Pterula sp., and
Mycena spp.

et al. 2002). No biological activities have been d) Aporpium caryae (Tremellaceae)

reported so far.
b) Stereum hirsutum (Stereaceae)
Scavengers of free radicals are considered as pos-
sible protectants of cells and tissues against oxi-
dative damage and resulting diseases. In a search
for new antioxidants Yun et al. (2002) isolated
sterins A and B (Fig. 10.14) from fermentations
of a strain of Stereum hirsutum collected in Korea.
Sterin A was found to inhibit lipid peroxidation in
an assay using rat liver microsomes at concentra-
tions of 8 mg/ml. In the same assay vitamin E was
ten times more effective.
c) Antrodia serialis (Polyporaceae s. lat.)
Serialynic acid (Fig. 10.14) was isolated from agar
cultures of Antrodia serialis in a screening for new
antifungal compounds (Kokubun et al. 2007).
Phenols with an isopentenyne side-chain like sic-
cayne (Kupka et al. 1981) are sometimes produced
by both ascomycetes and basidiomycetes. Serialynic
acid has been found to be weakly active against
some phytopathogenic fungi.
The aporpinones (Fig. 10.14), four furanones
containing an acetylene moiety, were isolated
from cultures of Aporpium caryae (Levy et al.
2003). Aporpinone B and its acetyl derivative
exhibited modest antibacterial activity against
Bacillus subtilis, Staphylococcus aureus, and
Escherichia coli.
e) Bondarzewia montana (Bondarzewiaceae)
When exposed to aqueous KOH the root of Bon-
darzewia montana turns bright yellow. The main
chromogen was isolated and turned out to be a
new compound which was named montadial
(Fig. 10.14). When tested for biological activity
montadial exhibited weak phytotoxic and stron-
ger cytotoxic activities, the latter starting from
5 mg/ml (Sontag et al. 1999).
f) Cortinarius sp. (Cortinariaceae)
Cortamidine oxide (Fig. 10.14), an interesting
asymmetrical disulfide metabolite, was isolated
from the fruiting bodies of a Cortinarius sp.
collected in New Zealand (Nicholas et al. 2001).
 Secondary Metabolites of Basidiomycetes 225

OH OH

OH OH

HO O HO O
O O
O O
O OH O O

Thelephantin A Thelephantin H
HO
OH OH

O O
OH OH
O O
O
O O O O
O
O
O O O
O O
O O O O
HO HO
Curtisian A HO Curtisian C
O
HO O OH

Thelephoric acid
HO O OH
O OR2
O
N
AcO OAc O
N+
R1O
O O- OH

HO OH

Sarcodonin R1 = Ac R2 = H
Sarcodonin d R1 = H R2 = CH3

Fig. 10.13. Terphenyls from Thelephora aurantiotincta, T. terrestris, Hydnellum caeruleum, Sarcodon leucopus,
S. scabrosus, and Paxillus curtisii

Cortamidine oxide has both significant antimicro- h) Serpula himantoides (Coniophoraceae)

bial and cytotoxic activity. Other compounds
containing a pyridine N-oxide functionality have
been reported from Cortinarius species before,
namely, the toxin orellanine.
g) Chamonixia pachydermis (Boletaceae)
An unusual oxalylated tetramic acid, pachyder-
min (Fig. 10.15), was isolated from the gastromy-
cete Chamonixia pachydermis collected in New
Zealand (Lang et al. 2006). When tested for anti-
bacterial activity pachydermin did not exhibit an-
tibiotic activity.
The himanimides (Fig. 10.15) were isolated from
fermentations of a Serpula himantoides strain col-
lected in Chile (Aqueveque et al. 2002). All four
compounds are succinimide and maleimide deri-
vatives, of which two are N-hydroxylated. Only
himanimide C exhibits broad antibacterial, anti-
fungal, and cytotoxic activity, suggesting a link to
the N-hydroxylated maleinimide moiety.
i) Pholiota spumosa (Strophariaceae)
(R)-2-hydroxyputrescine dicinnamamide and (2R)-
2-[(S)-3-hydroxy-3-methylglutaryloxy]putrescine-
dicinnamamide (Fig. 10.15), two new cinnamic
226 Anja Schüffler and Timm Anke

O H
O
O O
O O HO
OH

OH O
H O HO OH
Tremellin Sterin A Sterin B
OH
OH
OH
O
O O

OH
OH
Serialynic acid Aporpinone A

OH O O

OH
O O O
O O O
O
OH OH O
4'-Hydroxyaporpinone A Aporpinone B 1"-Acetylaporpinone B

O OH

S
HO O N+ S N N+
OH O- H O-
Montadial Cortamidine oxide

Fig. 10.14. Tremellin from Tremella aurantialba, sterins from Stereum hirsutum, serialynic acid from Antrodia serialis,
aporpinones from Aporpium caryae, montadial from Bondarzewia montana, and cortamidine oxide from Cortinarius sp.

acid were isolated from fruiting bodies of Pho-
liota spumosa in addition to the known mayte-
nine (N1,N8-dicinnamoyl spermidine) by
Clericuzio et al. (2007). Both compounds exhibit
modest cytotoxic activities. Unfortunately, the
second compound was named pholiotic acid, a
name earlier given to another (illudalane) me-
tabolite of P. destruens (Becker et al. 1994).
D. Amino Acid Derivatives
Basidiomycetes produce a number of cyclopep-
tides with very interesting biological activities.
These are being dealt with in Chap. 13.
a) Aporpium caryae (Tremellaceae)
A culture of Aporpium caryae yielded isolates of
1H-indole-3-carboxylic acid, 1-(1,1-dimethyl-2-
propenyl) methyl ester, and 1H-indole-3-carbox-
ylic acid, 1-(2,3-dihydroxy-1,1-dimethylpropyl)
methyl ester (Fig. 10.16), two metabolites of
mixed biogenetic origin (Levy et al. 2000). Both
metabolites exhibited modest antifungal activities
against Cladosporium cucumerinum.
III. Conclusions
As can be deduced from the numerous new struc-
tures, interest in basidiomycete secondary metab-
olism has gained momentum. The biological
activities are interesting and may help to define
new lead compounds offering structures not easily
detected by the random screening of libraries
derived from combinatorial chemical synthesis.
The availability of basidiomycete metabolites is
facilitated by important progress in fermentation
technologies and genetics, opening access to tem-
plates for chemical syntheses and providing new
chemical approaches to yet unexplored biological
targets.
 Secondary Metabolites of Basidiomycetes 227

OH Fig. 10.15. Himanimides, dicin-

namamides, and pachydermin
O O
OH from Serpula himantoides, Pho-
O O liota spumosa, and Chamonixia
pachydermis
HN HN

O O
Himanimide A Himanimide B

O O
O O

HO N HO N

O O
Himanimide C Himanimide D

O
H
N
N
H H
OH O

(R)-2-Hydroxyputrescine dicinnamamide
OH
O Cl
H
N
N
H H
O O O H
O N
OH
O
HO O OH
HO O
{(2R)-2-[(S)-3-Hydroxy-3-
methylglutaryloxy]putrescine dicinnamamide}
(pholiotic acid) Pachydermin

O O
O O

N N
OH
OH

1H-indole-3-carboxylic acid, 1-(1,1- 1H-indole-3-carboxylic acid, 1-(2,3- Fig. 10.16. Amino acid deriva-
dimethyl-2-propenyl) methyl ester dihydroxy-1,1-dimethylpropyl) methyl ester tives from Aporpium caryae
228 Anja Schüffler and Timm Anke
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11 Identification of Fungicide Targets in Pathogenic Fungi

ANDREW J. FOSTER1, ECKHARD THINES1

CONTENTS with regard to fungicide targets and available

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 fungicides and the outlook for the development
II. Currently Deployed Fungicides . . . . . . . . . . . . . . . . 233 of novel fungicides, with special focus on the ap-
III. What Are the Ideal Attributes plication of the ‘omics’ technologies in the drug
of the Fungicides of the Future? . . . . . . . . . . . . . . . 234 development process. The availability of these ge-
IV. The Role of Traditional Screening Approaches
Used in Fungicide Development . . . . . . . . . . . . . . . 234 nome-wide technologies represents a major new
V. Determining the Mode of Action opportunity to identify novel fungicide targets
of an Anti-Fungal Compound . . . . . . . . . . . . . . . . . . 235 within pathogenic fungi. Even within the best
VI. Genome-Wide Approaches . . . . . . . . . . . . . . . . . . . . . 235 studied pathogenic fungi there is still a great deal
A. Transcriptomics: Microarrays . . . . . . . . . . . . . 235 to learn about how disease is established and
B. Studying Genome-Wide Transcriptional
Changes Which Accompany maintained. A major challenge for drug develop-
Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 ment today is how best to employ the new genome-
C. Cross-Species Comparisons: Comparative wide technologies to indentify which metabolic
Genomic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 pathways and gene products are critical for disease
D. Identifying Possible Drug Targets by establishment and progression and thus to increase
Comparison of the Transcriptome of
Mutant and Wild-Type Cells . . . . . . . . . . . . . . . 239 the probability of finding novel fungicide tar-
E. Exploring Drug Resistance Using gets. This chapter therefore reviews genome-wide
Transcriptome Analysis: Candida approaches that have emerged in the post-genomics
albicans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 era, e.g. comparative genomics and gene expres-
F. MPSS and SAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 sion profiling experiments, including technologies
G. Transcriptomics: Outlook . . . . . . . . . . . . . . . . . . 242
H. Other ‘omics’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 such as microarray-based transcriptional profiling,
VII. Conclusions/Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 SAGE, and MPSS. It also reviews results of the
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 application of such projects to date in the field.

II. Currently Deployed Fungicides

I. Introduction
The frequent occurrence of resistance to commonly
Fungicides have become an extremely important deployed anti-fungals necessitates a continued
means to combat problems caused by fungal search for novel fungicides. Ideally new compounds
pathogens both within the agricultural and medic- should have a novel mode of action, have a minimal
inal fields. Although a number of very effective risk in terms of resistance development, and show a
fungicides have been developed, the emergence of high degree of specificity towards the target organ-
resistance has brought a constant need for the ism. These compounds must also satisfy the current
identification of new fungicide targets and for stringent regulations regarding ecotoxicity and user
novel agents which act against these targets. safety prior to registration.
This chapter discusses the current situation The most successful fungicides on the market
today act relatively broadly by targeting fungal
1 vegetative growth and thus the entire fungal life
Institut für Biotechnologie und Wirkstoff-Forschung e.V./
Institute for Biotechnology and Drug Research, Erwin-Schrödin- cycle. Examples of a successful mode of action
ger-Strasse 56, 67663 Kaiserslautern, Germany; e-mail: foster@ classes interfering with biochemical processes
ibwf.de, thines@ibwf.de essential in fungi include compounds targeting

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
234 Andrew J. Foster and Eckhard Thines
respiration and sterol biosynthesis. An example of
the former mode of action class is given by com-
pounds which target mitochondrial electron
transport within the respiration chain. Fungicides
of the strobilurin class have this mode of action.
These compounds are structurally based on natu-
ral products and interfere with the ubiquinol-
cytochrom C oxidoreductase of the cytochrome
bc1 in complex III (Becker et al. 1981; Anke and
Steglich 1999). A number of fungicides whose
mode of action is the inhibition of sterol biosyn-
thesis have been developed. The sterol biosynthet-
ic pathway includes several drugable targets, such
as a 3-keto reductase of an enzyme complex of the
sterol C-4 demethylation, the squalene epoxidase,
D14-reductase and/or D8!D7-isomerase, and ste-
rol 14a-demethylation, which is inhibited by azole
fungicides (Köller et al. 1992; Debieu et al. 2001).
Although both respiration and sterol biosynthesis
have proved good targets for which very success-
ful fungicides have been produced, resistance
against such fungicides develops rapidly resulting
in a high demand for new targets and fungicides.
Furthermore, several fungicides with a broad
range of activity have side-effects on benign fun-
gal species, animals, or plants which cannot be
excluded. For this reason there is a need for new
types of fungicides which exhibit novel modes of
action.
III. What Are the Ideal Attributes
of the Fungicides of the Future?
Novel targets can be located either in biochemical
or signalling pathways essential for vegetative
growth or for pathogenic development of the fun-
gus. Targets with high specificity can thereby be
expected in pathways involved in adhesion, host-
recognition/pre-penetration processes, host colo-
nisation, and the final reproductive differentiation
processes during pathogenic development. An at-
tractive proposition is the development of fungi-
cides interfering with pathogenic development
but not with vegetative growth. Such a strategy
could prevent or cure infections by fungal patho-
gens without affecting neutral or benign species.
Within the melanin biosynthetic pathway en-
zymes such as tetra- or trihydroxynaphthalene
reductase and sytalone dehydratase have been
identified as targets for non-fungitoxic fungicides.
Fungicides interfering with these targets, such as
tricyclazole, phthalide, pyrochilon, and capropa-
mid have been successfully used in plant protec-
tion (Nakasako et al. 1998; Thompson et al., 2000).
Such inhibitors of melanin biosynthesis prove
effective against the rice blast fungus Magna-
porthe grisea where penetration of this species is
prevented by blocking the formation of the
appressorial melanin layer required for building
turgor pressure within this cell (Howard and Fer-
rari 1989). A good degree of specificity for the
target organism is achieved in this case because
these inhibitors specifically target enzymes
involved in the production of melanin from a
pentaketide precursor while melanin in other
organisms uses alternative biosynthetic pathways
not affected by such agents (Thines et al. 2004).
The differentiation process of appressorium
formation itself may offer novel targets for mod-
ern plant protection strategies. Inhibitors interfer-
ing with the formation of infection structures
might be found which do not interfere with myce-
lial growth. One example for such a protective
fungicide is quinoxyfen, a compound which pre-
vents appressorium formation in Blumeria grami-
nis. The mode of action of quinoxyfen is still
unclear; however it was proposed recently that
quinoxyfen interferes with signalling (Wheeler
et al. 2003) and studies of quinoxyfen-resistant
mutants recently implicated inhibition of a serine
esterase (Lee et al. 2008).
The disadvantage of fungicides with high
specificity is their low range of application, which
makes them an unattractive commercial proposi-
tion for companies. Considering the high costs
incurred during the development and registration
of a novel fungicide, companies must have to
opportunity to recover the enormous costs of
development; fungicides active only against a nar-
row range of species are obviously much less likely
to bring a profit to the developer.
IV. The Role of Traditional Screening
Approaches Used in Fungicide
Development
In recent decades traditional screening approaches
with compounds derived from chemical synthesis
or natural products in greenhouses were very suc-
cessful and led to a generation of very effective
 Identification of Fungicide Targets in Pathogenic Fungi
fungicides with toxicologically favourable profiles.
Therefore screening on living organisms/plants
will play an important role in the future of plant
protection research. However, the drawback of
this traditional approach is that large numbers of
compounds must be tested in order to get to a hit.
To overcome this, target-based screening plat-
forms and virtual molecular modelling systems
have been developed in which large numbers of
molecules can be tested.
V. Determining the Mode of Action
of an Anti-Fungal Compound
Determining whether a new compound has a
novel mode of action is a critical step in the drug
development process and this must be achieved at
an early stage. To this end in vitro screening
systems targeting distinct biochemical reactions/
pathways can also be used to identify the mode of
action of compounds showing good antifungal
activity during in vivo screening experiments.
New compounds for which the mode of action is
not known can initially be assessed in simple
biochemical tests, such as inhibition of respiration
or sterol biosynthesis. Furthermore fungicide-re-
sistant mutants have successfully been used to
identify which protein is likely targeted by the
agent. When the mode of action of fungicidal
compounds cannot be identified by such ap-
proaches, novel methods such as metabolome,
proteome, or transcriptome analysis can provide
signature patterns which can be compared to pat-
terns obtained from experiments with known
compounds and may indicate whether or not it
is likely that the drug has a novel mode of action.
VI. Genome-Wide Approaches
Genome-wide technologies, e.g. gene expression
profiling and comparative genomics, lead to the
identification of a large number of potential can-
didate genes encoding factors essential either for
invasive growth and development or for vegeta-
tive growth. However, these potential targets have
to be validated by gene deletion or gene-silencing
experiments. Even if deletion of the gene is lethal
or the mutant shows a non-pathogenic phenotype,
235
it must be further established whether the target is
drugable in vivo.
Once a target has been validated, target-based
ultra-high throughput biochemical screening sys-
tems can be established in which hundreds of
thousands of compounds can be tested daily.
The challenge for modern agrochemical research
is to convert the in vitro activity of compounds
identified in such screening systems to products
active in vivo.
The post-genomics era is now truly upon us.
More than 50 different fungal genome sequences
are now publicly available and more fungal
sequencing projects are either planned or in prog-
ress. More than half of the finished genomes are of
pathogenic species and cover the most important
fungal pathogens of humans as well as many of the
most devastating fungal pathogens of commer-
cially important crops (Table 11.1). These
resources open the door to genome-wide analysis
within these species. Recently developed technol-
ogies allow the analysis of mRNA (transcrip-
tomics), protein (proteomics), and metabolite
(metabolomics) profiles. We focus here largely
on transcriptomics and proteomics.
A. Transcriptomics: Microarrays
Microarrays were first employed within fungi
more than ten years ago (DeRisi et al. 1997) and
microarray technology has since been widely used
to study a broad array of processes within fungi
(reviewed by Breakspear and Momany 2007). The
technology itself is a logical development from
Southern/Northern blotting and, very basically,
the microarray itself is a series of spots of nucleic
acids (cDNAs or oligonucleotides) which are
known as features or probes and which are at-
tached to a solid support. cDNA or cRNA samples
(somewhat confusingly these are known as targets
in this context) can then be hybridised to the
microarray and, following high stringency washes,
the degree of probe to target annealing at each
spot can be quantified and, by comparison of the
signal under different conditions, the relative
abundance of the target within two different sam-
ples can be assessed. Microarray technology is
described in detail elsewhere (e.g. see Heller
2002). Several applications of microarray technol-
ogy have emerged in recent years; however
here we confine our discussion largely to the
236 Andrew J. Foster and Eckhard Thines

Table 11.1. Fungal species whose genome sequence has been determined or for which a sequence release is imminent.
The latest status for ongoing projects and links to finished genomes are available at http://fungalgenomes.org/wiki/
Fungal_Genome_Links

Species Division Important features of the fungus Responsible body

Agaricus bisporus Basidiomycota Common edible button or table Joint Genome Institute
mushroom
Alternaria Ascomycota Plant pathogen (Brassica dark leaf WUSTL (access also via Broad Institute)
brassicicola spot of Brassica species)
Amanita bisporigera Basidiomycota Common deadly mushroom Michigan State University
(Destroying Angel)
Ashbya gossypii Ascomycota Plant pathogen (cotton) Broad Institute
Aspergillus fumigatus Ascomycota Human pathogen, especially in the Sanger Centre/TIGR
immunocompromised
A. oryzae Ascomycota A species widely used in National Institute of Technology and
biotechnology Evaluation
A. nidulans Ascomycota A model filamentous fungus; Broad Institute
widely studied
A. terreus Ascomycota Opportunistic human pathogen Broad Institute
A. niger Ascomycota Common contaminant of food Joint Genome Institute
A. clavatus Ascomycota A rare opportunistic human TIGR
pathogen
Batrachochytrium Chytridiomycota Causes the amphibian disease Broad Institute/Joint Genome Institute
dendrobatidis chytridiomycosis (two different strains sequenced)
Botrytis cinerea Ascomycota Plant pathogen with broad host Broad Institute
range
Candida albicans Ascomycota Opportunistic human pathogen; Broad Institute
dimorphic yeast
C. dubliniensis Ascomycota Opportunistic human pathogen; Sanger Centre
close relative of C. albicans
C. glabrata Ascomycota Opportunistic pathogen (non- Sanger (finishing), Génolevures
dimorphic)
C. guilliermondii Ascomycota A xylitol producer Broad/Génolevures
C. lusitaniae Ascomycota Opportunistic pathogen Broad Institute
(amphotericin B-resistant)
C. parapsilosis Ascomycota Opportunistic human pathogen; Sanger
forms biofilms
C. tropicalis Ascomycota Opportunistic human pathogen Broad Institute
Chaetomium Ascomycota Causes infections of human nails Broad Institute
globosum and skin; allergen
Coccidioides immitis Ascomycota Human pathogen; causes Valley Broad Institute (four different strains
fever sequenced)
C. posadsii Ascomycota Human pathogen Broad Institute
Colletotrichum Ascomycota Plant pathogen (causes Broad Institute (still in progress)
graminicola anthracnose diseases of
cereals)
Coprinus cinereus Basidiomycota Model basidiomycete ‘ink cap’ Broad Institute/Duke University
fungus
Cryptococcus Basidiomycota Human pathogen; serotypes A and Broad Institute
neoformansa B have been sequenced
C. gattii Basidiomycota Human pathogen (cryptococcosis) Broad/British Columbia Genome
Sequencing Centre
Debaryomyces Ascomycota A cryotolerant, marine yeast Génolevures
hansenii
Fusarium Ascomycota Plant pathogen (wheat head Broad Institute
graminearum blight)
F. oxysporum Ascomycota Plant pathogen (wilt diseases of Broad Institute
many species of plants)
F. verticillioides Ascomycota Plant pathogen (‘foolish seedling’ Broad Institute
disease of rice seedlings)
(continued)
 Identification of Fungicide Targets in Pathogenic Fungi 237

Table 11.1. (continued)

Species Division Important features of the fungus Responsible body

Heterobasidion Basidiomycete Plant pathogen; causes root and Joint Genome Institute
annosum butt rot to conifers
Histoplasma Ascomycota Causes histoplasmosis; NAm1 and Broad Institute
capsulatum G186AR strains
Kluyveromyces lactis Ascomycota Industrial and model yeast species Génolevures
K. marxianus Ascomycota Yeast species/commercial Génolevures
producer of lactase
K. thermotolerans Ascomycota Yeast species associated with fruits Génolevures
K. waltii Ascomycota Yeast species Génolevures
Lacazia loboi Ascomycota Causal agent of lobomycosis Broad Institute
(human skin disease)
Laccaria bicolor Basidiomycota Ectomycorrhizal fungus Joint Genome Institute
Lodderomyces Ascomycota Closely related to Candida Broad Institute
elongisporus parapsilosis
Magnaporthe grisea Ascomycota Plant pathogen (rice blast); model Broad Institute
cereal pathogen
Malassezia globosa Basidiomycota Associated with dandruff Commercial (available through NCBI)
M. restricta Basidiomycota Associated with dandruff Commercial (available through NCBI)
Microsporum Ascomycota Human pathogen Broad Institute
gypseum (Dermatophytes)
Moniliophthora Basidiomycota Plant pathogen (causes cacao Unicamp
perniciosa disease)
Mycosphaerella Ascomycota Plant pathogen (banana Black Joint Genome Institute
fijiensis Sigatoka)
M. graminicola Ascomycota Plant pathogen (Septoria tritici Joint Genome Institute
blotch of wheat)
Nectria Ascomycota Human and plant pathogen Joint Genome Institute
haematococca
Neosartorya fischeri Ascomycota Aspergillus species causes food TIGR
spoilage/pathogenic (rare)
Neurospora crassa Ascomycota Model filamentous fungus for Broad Institute
basic research
Paracoccidioides Ascomycota Dimorphic; causal agent of Broad Institute (strains Pb01, Pb03, Pb18)
brasiliensis paracoccidioidomycosis
Paxillus involutus Basidiomycota Common ectomycorrhizal fungus Joint Genome Institute
Penicillium marneffei Ascomycota Human pathogen/thermally TIGR
dimorphic species
P. chrysogenum Ascomycota Industrially important source of Commercial (available through NCBI)
several b-lactam antibiotics
Phanerochaete Basidiomycota White rot fungus, used for Joint Genome Institute
chrysosporium bioremediation
P. carnosa Basidiomycota White rot fungus Joint Genome Institute
Pichia angusta Ascomycota Methylotrophic yeast species with Génolevures
biotechnological uses
P. stipitis Ascomycota Yeast species; used for xylose Joint Genome Institute/Génolevures
fermentation
P. farinosa Ascomycota Yeast species; emerging human Génolevures
(sorbitophila) pathogen
Phycomyces Zygomycota Model zygomycete; widely studied Joint Genome Institute
blakesleeanus
Phytophthora Oomycete Plant pathogen; potato blight Sanger Centre
infestans
Postia placenta Basidiomycota Wood-rooting fungus; causes Joint Genome Institute
brown rot
Puccinia graminis Basidiomycota Plant pathogen; rusts of cereals Broad Institute
Pyrenophora tritici- Ascomycota Plant pathogen; yellow leaf spot Broad Institute
repentis of cereals and grasses
Rhizopus oryzae Zygomycota Human pathogen Broad Institute
(continued)
238 Andrew J. Foster and Eckhard Thines

Table 11.1. (continued)

Species Division Important features of the fungus Responsible body

Sclerotinia Ascomycota Plant pathogen (root, crown, stem Broad Institute
sclerotiorum rots; varied hosts)
Saccharomyces Ascomycota Baker’s yeast; a leading model Broad Institute and others (several strains
cerevisiaeb organism in research sequenced)
S. bayanus Ascomycota Yeast species; used in wine making Broad Institute
S. castellii Ascomycota Yeast species WUSTL
S. kluyveri Ascomycota Yeast species WUSTL
S. kudriavzevii Ascomycota Yeast species WUSTL/Stanford
S. mikatae Ascomycota Yeast species Broad Institute/Stanford
S. paradoxus Ascomycota Yeast species Broad Institute/Stanford
Schizophyllum Basidiomycota Common fungus; causes white rot Joint Genome Institute I (near
commune completion)
Schizosaccharomyces Ascomycota Fission yeast species; grows Broad Institute
octosporus filamentously
S. japonicus Ascomycota Fission yeast species Broad Institute
S. pombe Ascomycota Fission yeast, very widely studied Sanger Centre
model fungus
Stagonospora Ascomycota Plant pathogen, blotch disease of Broad Institute
nodorum wheat
Trichoderma reesei Ascomycota Industrially important fungus Joint Genome Institute
T. virens Ascomycota Used in biocontrol against fungal Joint Genome Institute
pathogens
Uncinocarpus reesii Ascomycota A close relative of Coccidioides Broad Institute
Ustilago maydis Basidiomycota Plant pathogen (corn smut Broad Institute
disease); model organism
Verticillium dahliae Ascomycota Cause of wilt diseases in important Broad Institute
crop species
V. albo-atrum Ascomycota Cause of wilt diseases in important Broad Institute
crop species
a
Three different strains of Cryptococcus neoformans have been sequenced: C. neoformans var. neoformans JEC21 (TIGR), C. neoformans
var. neoformans B-3501A (Stanford University) and C. neoformans var. grubbii H99 (Duke/FGI)
b
Several strains of Saccharomyces cerevisiae have been sequenced or are being sequenced. For further details, see http://www.sanger.ac.
uk/Teams/Team71/durbin/sgrp/index.shtml

application of microarrays to the study of tran-
script abundance and how microarray analysis
might be exploited to assist in the identification
of novel fungicide targets. A number of different
examples to illustrate the different potential appli-
cations of microarrays are given below.
B. Studying Genome-Wide Transcriptional
Changes Which Accompany Differentiation
Within plant pathogenic fungi, early studies used
sub-genome-wide technologies, often derived
from the results of EST sequencing projects, to
study development (See Breakspear and Momany
2007 and references therein). In investigations of
the rice blast fungus Magnaporthe grisea, for ex-
ample, the first microarray experiments used
cDNA-based technology to investigate the relative
transcript abundance of 3500 features (cDNAs
with some redundancy) during infection-related
morphogenesis to investigate appressorium for-
mation (Takano et al. 2003).
A whole genome microarray of M. grisea is
now commercially available from Agilent Bio-
technologies and has been used to identify genes
important for appressorium formation (Oh et al.
2008). Genes responding to nitrogen starvation, a
situation believed to mimic the nutritional state of
the fungus post-penetration and which is purport-
ed to induce infection-related development, have
also been studied using these commercially avail-
able microarrays (Donofrio et al. 2006). One gene
which responds to nitrogen starvation, SPM1, is
predicted to encode a vacuolar serine protease. It
was deleted and shown to be required for normal
levels of virulence (Donofrio et al. 2006). Novel
gene products which are critical for appressorium
 Identification of Fungicide Targets in Pathogenic Fungi
formation have been uncovered by microarray-
based analyses, including a subtilisin-like prote-
ase and a NAD-specific glutamate dehydrogenase
(Oh et al. 2008). These studies clearly illustrate the
potential of genome-wide technologies to potenti-
ate the identification of potential new fungicide
targets by narrowing the search for possible path-
ogenicity factors.
In Fusarium graminearum, a whole genome
microarray has been used to investigate which
pathways are induced during the germination of
conidia, an important first step in the establish-
ment of disease (Seong et al. 2008). Clearly some
of these differentially regulated genes might prove
attractive as targets for drug intervention, although
obviously with such an analysis significant follow-
up experiments would be required in order to
identify which genes are essential for the process
studied, as these are likely only a small subset of the
total number identified.
Further commercially produced whole-ge-
nome microarrays are in development for other
plant pathogenic fungi, including Botrytis cinerea
and Mycosphaerella graminicola. It can be antici-
pated that the range of microarrays available and
their application within plant pathology will ex-
pand greatly in the future. Whether the applica-
tion of these microarrays will eventually reveal
fungicide targets which are conserved across a
broad range of species is open to question; how-
ever there is no doubt about the potential of this
technology to generate new experimental hypoth-
esis which could greatly facilitate the identifica-
tion of novel targets for drug control within
individual species.
Extensive microarray-based analysis can also
proceed in the absence of a whole-genome se-
quence. An example is the study of Both and co-
workers (2005), who looked at more than 2000
Blumeria graminis genes from an infection time-
course and revealed how several genes previously
implicated in disease progression as well as many
new unknown genes were expressed during infec-
tion-related development. Some of the genes iden-
tified during this study might be potential
candidates for fungicide targets, although proving
this is not straightforward in an obligate pathogen
such as Blumeria graminis.
The application of microarrays to the study of
fungal pathogens of humans is also becoming
increasingly commonplace. For example, Histo-
plasma capsulatum is an ascomycete dimorphic
239
fungus responsible for the human disease histo-
plasmosis. Microarray analysis using genomic
DNA-derived targets has identified genes specifi-
cally expressed in the pathogenic yeast form
(Hwang et al. 2003). Again significant follow-up
experiments are required to test whether any of
the products of these genes are essential for viru-
lence but, in terms of identifying potential novel
targets, such studies offer valuable new hypoth-
eses which will no doubt lead to the identification
of novel drug targets which could prove valuable
in future years.
C. Cross-Species Comparisons:
Comparative Genomic
Where significant similarity exists between closely
related fungal species there is a possibility to con-
duct cross-species comparisons using microar-
rays. A good example of such an application is
provided by the study of Moran and co-workers
(2004) who exploited Candida albicans-based ge-
nomic microarrays to compare the genome of this
species to the very closely related but less virulent
species C. dubliniensis. Although the vast majority
of genes were found to be highly conserved, sev-
eral hundred genes were identified which were
either absent in C. dubliniensis or whose sequence
was poorly conserved between these two species.
This study not only generated several testable
hypotheses which might shed light on the differ-
ence in virulence between these two species but
also provided a great deal of information about
the C. dubliniensis gene repertoire in the absence
of a genome sequence. Interestingly several of the
genes poorly conserved between the two species
studied are predicted to encode transcription fac-
tors and it would therefore be of significant inter-
est to extend these analyses by comparing the
transcriptome of the two species.
D. Identifying Possible Drug Targets
by Comparison of the Transcriptome
of Mutant and Wild-Type Cells
Using whole-genome microarrays to study a major
developmental switch such as germination or ap-
pressorium formation, several hundred to
thousands of genes have been found to be differen-
tially expressed (e.g. Oh et al. 2008; Seong et al.
2008). An alternative route to identify the genes
240 Andrew J. Foster and Eckhard Thines
involved in a particular process is to examine a
mutant blocked at that developmental stage. With
a view to drug development, this may be a more
attractive route, as the number of target genes
identified by this approach is more likely in the
tens rather than in the hundreds. Such an applica-
tion of microarrays is nicely illustrated by a recent
study of the human pathogen Cryptococcus neofor-
mans (Cramer et al. 2006). These workers identified
genes which are transcriptionally down-regulated
in a mutant which lacks a component of the cAMP
signalling pathway which is required for capsule
formation, a critical determinant of virulence. One
of these genes was found to encode a transcription
factor with similarity to yeast transcription factor
Nrg1p. A C. neoformans strain lacking this tran-
scription factor was generated and was also found
to be deficient in capsule formation. Microarrays
were then used to identify genes transcriptionally
dependent on the product of NRG1 and among the
71 genes identified was UGD1, a gene whose prod-
uct was previously implicated in capsule formation
in this fungus (Moyrand and Janbon 2004).
Although this gene had previously been identified,
this study illustrates the potential of the combina-
tion of microarray analysis and specific mutations
to uncover potentially drugable targets. In a similar
manner, Odenbach and co-workers (2007) used
microarrays to identify approximately 100 Magna-
porthe grisea genes whose transcription depends
on the Con7p transcription factor during germina-
tion of spores of this fungus. These included a
chitin synthase encoding gene CHS7 which, like
the con7 mutant, is affected in the formation of
the appressoria (Odenbach et al. 2007, 2009). Again
chitin synthase is already considered a potential
target for drug intervention in human disease, so
although this is not a novel fungicide target, this
study nicely illustrates the potential of using micro-
arrays in combination with specific mutants to
identify potential drug targets. In a similar manner
using a human pathogenic fungus, Ngugen and Sil
(2008) identified a Histoplasma capsulatum regula-
tor Ryp1p which is required for yeast phase gene
expression and identified several genes controlled
by this transcription factor by comparing tran-
script profiles of mutant and wild-type cells.
Identification of novel fungicide targets is
ultimately a process of elimination. Eliminating
which factors are not good targets from the likely
comparatively small number which are useful
(drugable) targets is an important step in target
identification. Mutant generation will continue to
be a decisive experiment in this process for years
to come. Any means by which it is possible to
narrow the search for new targets is obviously
attractive. Much research in the past few decades
in the academic world has focused on signal trans-
duction and, although it is questionable whether
any of the proteins identified by such studies are
themselves good targets, the mutants generated by
such studies could be exploited to identify down-
stream components of which a small subset are
likely to be essential for the process and which
may include useful targets for drug intervention.
E. Exploring Drug Resistance Using
Transcriptome Analysis: Candida albicans
A further application for microarrays within the
field of drug development is in the study of resis-
tance to fungicides. Such an approach has already
been adopted by a number of groups attempting
to understand the transcriptional response to fun-
gicide treatment. In this respect by far the best
studied species within fungi is the opportunistic
fungal pathogen of humans Candida albicans.
Two of the most commonly deployed drugs in
the treatment of diseases caused by C. albicans
are the polyene fungicide amphotericin B (AMB)
and fungistatic azole compounds such as flucona-
zole. Although the mode of action of these drugs
differs, resistance to both drugs within clinical
isolates is often associated with induction of
drug efflux transporter activity and/or alterations
to the ergosterol biosynthetic enzymes (reviewed
by Akins 2005). This is not surprising, as both
drugs are thought to ultimately impact on mem-
brane integrity. A broader understanding of the
resistance mechanisms to these and other drugs
has come from several microarray-based studies
which have examined transcript profiles within
amphotericin B-resistant isolates or drug-treated
wild-type strains. These studies both confirmed
previous reports which suggested induction of
drug efflux transporter activity and/or alterations
to the ergosterol biosynthetic enzymes and also
led to the identification of further genes which are
induced specifically on treatment with drugs of a
specific class or which seem to be part of a more
general response to drug challenge. Several genes
whose transcription is altered by drug treatment
 Identification of Fungicide Targets in Pathogenic Fungi
are otherwise uncharacterised and their participa-
tion in drug response may therefore be the first
clue to their function. Furthermore the specific
transcript profile induced by treatment with a
particular drug might also be used as a ‘finger-
print’ for drugs of that type and might assist in
assigning a tentative mode of action to a drug
whose targets is unknown or unclear. C. albicans
is also known to commonly form biofilms which
show increased resistance to commonly deployed
antifugals (Chandra et al. 2001; Ramage et al.
2001). Farnesol, a quorum-sensing molecule, was
shown to prevent biofilm at high concentrations
and is considered a possible novel Candida con-
trol agent (Ramage et al. 2002). Microarray analy-
sis subsequently shed some light on the mode of
action which was previously not known, suggest-
ing that the compound might in part prevent fila-
mentous growth by inducing the transcription of
the TUP gene which encodes a repressor of hyphal
development (Cao et al. 2005). Again this study
highlights the utility of microarray experiments in
understanding drug response and mechanisms of
resistance.
The response to azole fungicides has also been
studied in Aspergillus fumigatus where, as is the
case for C. albicans, resistance to clinically deployed
drugs of this class is increasingly problematic.
Several thousand genes were found to be differen-
tially regulated in response to voriconazole (da
Silva Ferreira et al. 2006). Up-regulated genes in-
cluded several transporters of ABC and MFS type
which might play a role in drug efflux.
The use of microarrays for the study of resis-
tance to drug treatment can be expected to lead to
valuable insights into what are the critical deter-
minants of resistance and increasing application
in this field as well as in determination of mode of
action is anticipated in the future.
F. MPSS and SAGE
Serial analysis of gene expression (SAGE; Velcu-
lescu et al. 1995) and massively parallel signature
sequencing (MPSS; Brenner et al. 2000) are related
technologies which yield short sequence signa-
tures derived from mRNAs. By comparison of
the relative abundance of these signatures within
libraries derived from different mRNA popula-
tions, a semi-quantitative measure of transcript
abundance in a sample is obtained.
241
Examples of the application of these technol-
ogies are studies conducted using the plant patho-
gens Magnaporthe grisea (Irie at al. 2003) and
Blumeria graminis (Thomas et al. 2002). Several
genes dependent on cAMP were revealed using
SAGE in Magnaporthe, including a number
which have already been shown to be essential
for pathogenicity (Irie et al. 2003). In B. graminis,
an obligate pathogen, several thousand SAGE tags
were sequenced and more than 100 genes differ-
ential transcribed during infection-related devel-
opment were highlighted (Thomas et al. 2002).
This study highlights one great advantage of
these technologies: they can be applied to any
organism irrespective of whether a genome se-
quence is available or not. A further application
for such technologies is in determining which of
the predicted genes within an annotated genome
is really transcribed. Thus in the case of M. grisea,
a large number of the genes predicted to exist in
the genome by automated annotation were subse-
quently shown to be really transcribed using SAGE/
MPSS (Gowda et al. 2006). In fact the same study
indicted the existence of several thousand addition-
al transcripts which do not correspond to any
annotated gene and has therefore expanded the
M. grisea gene repertoire.
SAGE has also been used to study protein ki-
nase A (PKA)-dependent gene expression in the
corn smut pathogen Ustilago maydis (Larraya
et al. 2005). By analysis of transcript profiles
based on the sequences of at least 40 000 tags for
the wild type and two mutants in the PKA pathway,
the authors were able demonstrate novel functions
for PKA signalling in this organism, including a
link to phosphate metabolism. Because PKA sig-
nalling is critical for virulence in this species, such
analyses pinpoint downstream targets of PKA
signalling which may well include potential new
targets for fungicide control of corn smut.
Within fungi which are pathogenic to humans,
SAGE has been employed for transcriptome anal-
ysis most extensively in Cryptococcus neoformans.
One comparison used 49 224 SAGE tags from each
of two protein kinase A (PKA) pathway mutants
and a wild-type control under conditions known
to induce capsule formation in this species (Hu
et al. 2007). Among the 599 tags exhibiting altered
abundance between these SAGE libraries were
several corresponding to genes whose products
might play a role in secretion. Furthermore,
using inhibitors of secretion, the authors were
242 Andrew J. Foster and Eckhard Thines
able to demonstrate that capsule formation is
inhibited at drug concentrations which do not
affect growth in culture. This indicates that, as
one might anticipate, secretion plays an important
role in capsule formation. These results not only
expand the understanding of the targets of PKA
signalling but also highlight the potential of tar-
geting components of the secretion machinery in
order to control this species.
In conclusion SAGE and MPSS are powerful
means to obtain a semi-quantitative assessment of
relative transcript abundance under different con-
ditions. There is no doubting the value of these
technologies in providing information which
could suggest which pathways or enzymes are
likely to be activated under specific conditions.
Additionally these technologies have been suc-
cessfully employed in fungal species which have
not yet been completely sequenced. As with
microarrays, these technologies could readily be
employed to suggest possible new targets for
drug intervention; however, in comparison to
microarrays, MPSS and SAGE have been much
less broadly applied experimentally and it is there-
fore probable that their application in future drug
development will also be peripheral.
G. Transcriptomics: Outlook
Microarrays and related technologies have unques-
tionably provided the researcher aiming to study
plant–pathogen interactions with powerful tools
which is are now very broadly accepted and in-
creasingly widely used. It is likely that with ever
improving technologies and decreasing prices such
forms of analysis will become available to most
researchers in the coming years. Nevertheless it is
important to be aware that transcript abundance
does not always correlate well with protein abun-
dance. It has been common to extrapolate from the
results of such analyses and suggest that certain
pathways are activated in response to certain de-
velopmental or environmental conditions or as a
result of certain mutations. Although this is likely
to be true in the majority of cases, these technolo-
gies should at best be viewed as a very elegant
means to generate hypotheses in the absence of
any preconceptions of the experimental outcome.
Although it is easy to be blinded by the power of
this technology, any study which reports only
microarray data is likely to provide very many
new questions but no real answers. The real power
of genome wide analysis of transcript abundance
will only be realised in combination with other
approaches which address the response of the or-
ganism at other levels and by genetic manipula-
tions to address the question as to whether the
observed alteration in transcript abundance has
any meaningful consequences for the test organ-
ism. Genetic manipulation of most fungal patho-
gens remains the major bottleneck in moving from
the results of genome-wide studies of transcript
abundance to establishing that the product of a
particular gene is really important or essential in
establishing disease (and therefore a potential drug
target).
H. Other ‘omics’
The ultimate targets of most known fungicides are
proteins and there is some logic to working direct-
ly at the level of the protein. Additionally there are
many examples where the major level of control
which determines protein abundance is post-tran-
scriptional. For these reasons proteome profiling
represents an attractive approach to identify po-
tential new fungicide targets. Proteome profiling
studies in Aspergillus species have been used to
identify proteins expressed during various physi-
ological states of the cell (for a review, see Kim
et al. 2008). Especially in the opportunistic human
pathogenic fungus Aspergillus fumigatus, compar-
ative proteome studies have been conducted in
order to identify proteins involved in virulence
or essential for mycelial growth that may serve
as targets for antimycotic therapies. Bruneau and
co-workers focussed on surface proteins thereby
identifying glycosylphosphatidylinositol-anchored
membrane proteins which had previously been
shown to be involved in cell wall biosynthesis
(Mouyna et al. 2000). It was shown that, out of
five proteins with unknown function, the Ecm33
protein influences the conidial cell wall biosynthe-
sis/cell wall morphogenesis (Chabane et al. 2006).
A proteome study conducted by Asif and co-
workers (2006) led to the identification of 26 co-
nidial cell surface proteins as potential vaccine
candidates. Several of the proteins identified had
no known function and may therefore be novel
targets for therapeutic approaches.
In proteome studies using mutants it was re-
cently shown that enhanced PKA activity results in
 Identification of Fungicide Targets in Pathogenic Fungi
the activation of stress-associated proteins, enzy-
mes involved in protein biosynthesis, and glucose
catabolism. Enzymes involved in nucleotide and
amino acid biosynthesis and enzymes involved in
catabolism other than glucose were down-regu-
lated by PKA signalling (Große et al. 2008). It was
furthermore found that septin and b-tubulin were
down-regulated by enhanced PKA signalling, indi-
cating that cAMP/PKA signalling is involved in the
regulation of fungal morphogenesis and may there-
fore be of relevance for virulence. These findings
are backed by mutant studies showing the rele-
vance of PKA/cAMP signalling in Aspergillus fumi-
gatus (Liebmann et al. 2004).
Proteomic analysis has also been carried out
in the fungal human pathogen Candida glabrata,
where a recent study using mutants lacking the
Ace2 transcription factor which have an increased
ability to cause disease. The types of proteins
identified suggested that this factor controls the
transcription of genes involved in cell wall biogen-
esis in this species (Stead et al. 2005).
In conclusion proteome analysis must been
regarded as powerful tool to identify proteins re-
quired for structural integrity of the cells or for
pathogenicity and it is likely that this approach
might find application in the search for suitable
targets for therapeutic approaches.
VII. Conclusions/Outlook
Genome-wide studies typically yield large numbers
of candidate genes of which it is likely that only a
small subset will be essential for the process stud-
ied. So for example, of several genes identified as
differentially regulated during infection-related
morphogenesis in Magnaporthe grisea only three
out of 16 selected for mutation were found to be
essential for pathogenicity. In order to fully exploit
the results of genome-wide analysis there is a clear
need for streamlined strategies for high-through-
put analysis of gene function. Given that a subset of
genes may be essential for viability, an attractive
approach to investigate whether a particular
gene represent a useful fungicide target would be
gene silencing because inducible gene silencing
might then allow the analysis of essential genes.
Again taking Magnaporthe as an example, gene
silencing has been shown to be effective in this
fungus (Kadotani et al. 2003) and tools for
243
high-throughput analysis using this technology
show great promise for the future (Nguyen et al.
2008). A collection of mutants deleted for all of the
predicted genes, as has been developed for Saccha-
romyces cerevisiae, is an attractive resource; how-
ever the generation of such a resource is a major
undertaking and it is likely that the intelligent use
of transcription profiling combined with mutation
of a subset of likely candidates is a more economical
way to identify novel fungicide targets. Although
the targets of most of the currently deployed fungi-
cides were discovered by more traditional
approaches, it is important to remember that ge-
nome-wide technologies are relatively new and that
it may be several years before they yield their first
fruits in terms of identification of new drug targets.
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12 Helminth Electron Transport Inhibitors Produced by Fungi

ROKURO MASUMA1, KAZURO SHIOMI1, SATOSHI ŌMURA1

CONTENTS phosphorylation (Saraste 1999). Electrons gener-

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 ated from NADH and FADH2 pass through com-
II. Inhibitors of Complex I . . . . . . . . . . . . . . . . . . . . . . . . . 250 plexes I–IV to produce a proton gradient, which is
III. Inhibitors of Helminth Complex I . . . . . . . . . . . . . 251 harnessed by complex V (Fig. 12.1).
A. NADH-Fumarate Reductase . . . . . . . . . . . . . . . . 251 Inhibitors of electron transport and oxidative
B. Nafuredin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
1. Producing Strain and Fermentation . . 251 phosphorylation enzymes are used to study the
2. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 mechanism of energy conversion. Some of them
3. Enzyme Inhibition and Biological have been developed into antifungal, insecticidal,
Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 antiparasite and other anti-infectious agents. For
4. Nafuredin-g and its Analogs . . . . . . . . . . . 254 example, carboxin (1, complex II inhibitor) and
C. Paecilaminol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
D. Verticipyrone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 azoxystrobin (2, complex III inhibitor) are used
E. Ukulactones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 against plant-pathogenic fungi, fenpyroximate (3,
IV. Inhibitors of Complex II . . . . . . . . . . . . . . . . . . . . . . . . 258 complex I inhibitor) and chlorfenapyr (4, uncou-
A. Atpenins and Harzianopyridone . . . . . . . . . . . 258 pler) are used to combat insects or acari, and
1. Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 bithionol (5, complex II inhibitor) and atova-
2. Enzyme Inhibition and Biological
Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 quone (6, complex III inhibitor) are used against
B. Other Complex II Inhibitors . . . . . . . . . . . . . . . 261 various parasites (Fig. 12.2). Though they are syn-
V. Other Electron Transport Inhibitors . . . . . . . . . . 262 thetic compounds, several are derived from natu-
A. Inhibitors of Complex III . . . . . . . . . . . . . . . . . . 262 ral compounds. Compound 2 is an analog of
B. Inhibitors of Complex V . . . . . . . . . . . . . . . . . . . . 263 strobilurin A (Fig. 12.3, 7) produced by a basidio-
C. Uncouplers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 mycete (Sauter et al. 1999), and the origin of 6 is
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 dioxapyrrolomycin (Fig. 12.3, 8), produced by
Streptomyces spp. (Addor et al. 1992).
Many electron transport inhibitors and oxida-
tive phosphorylation enzyme inhibitors have been
I. Introduction isolated from natural origins (Lardy 1980; Degli
Esposti 1998; Ueki et al. 2000). A famous complex
I inhibitor, rotenone (Fig. 12.4, 9), is a plant me-
The electron transport chain is present in mito-
tabolite and used effectively as an insecticide.
chondria (eukaryotes) or plasma membrane
Piericidin A (Fig. 12.4, 10) and antimycin A3a
(prokaryotes) and is linked with oxidative phos-
(Fig. 12.4, 11) are produced by Streptomyces spp.
phorylation to produce ATP. The chain consists of
and widely used as complex I and complex III
complex I (NADH-ubiquinone reductase), com-
inhibitors respectively. Siccanin (Fig. 12.4, 12),
plex II (succinate-ubiquinone reductase), complex
also produced by a fungus, inhibits complex II
III (ubiquinol–cytochrome-c reductase, cyto-
and is used against surface mycosis.
chrome bc1 complex), complex IV (cytochrome-c
During the course of screening for anthel-
oxidase) and complex V (ATP synthase, FOF1-
mintic antibiotics, some new compounds isolated
ATPase) which culminates in ATP via oxidative
from the culture broths of fungi were found to
be NADH-fumarate reductase (NFRD) inhibi-
1
The Kitasato Institute for Life Sciences and Graduate School of tors (Shiomi and Ōmura 2004; Kita et al. 2007;
Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Ōmura and Shiomi 2007). Among them, some
Minato-ku, Tokyo 108-8641, Japan; e-mail: masuma@lisci.kita-
sato-u.ac.jp, shiomi@lisci.kitasato-u.ac.jp
compounds show specific inhibition against

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
248 Rokuro Masuma et al.

Fig. 12.1. Electron transport and oxidative phosphorylation in mitochondria

O CH3
CH3
N N H3C O
O CH3 CH3
H O N O
O N
S N
CN O OCH3 N O
O H3C
OCH3

Carboxin (1) Azoxystrobin (2) Fenpyroximate (3)

N Cl
Br OH OH
Cl S Cl O
F3C N
Cl
O
Cl Cl OH
H3C O

Chlorf enapyr (4) Bithionol (5) Atovaquone (6)

Fig. 12.2. Structures of practically used electron transport inhibitors

NO2
Fig. 12.3. Structures of strobi- Cl
CH3
lurin A and dioxapyrrolomycin Cl

O Cl N H
OCH3 H

OCH3 O
O Cl

Strobilurin A (7) Dioxapyrrolomycin (8)

 Helminth Electron Transport Inhibitors Produced by Fungi 249

H CH2
CH3 CH3 CH3 CH3
H O CH3
O H3CO N CH3
O
OH
H H3CO CH3
O OH
H3CO
OCH3

Rotenone (9) Piericidin A (10)

O
H CH3 O OH O
O CH3
OHC N N
CH3
H O CH3 H H
OH O
H3 C O CH3 H3C O
CH3 CH3
O

Antimycin A3a(11) Siccanin (12)

Fig. 12.4. Popular natural electron transport inhibitors

Table 12.1. Electron transport and oxidative phosphorylation inhibitors produced by fungi

Compound Producer Biological activity

Complex I inhibitors
Cochlioquinones A (16) and B (17) Cochliobolus miyabeanus Anthelmintic, phytotoxic, etc.
Isocochlioquinone A (18) Bipolaris bicolor Phytotoxic
Stemphone A (19) Stemphylium sarcinaeforme Phytotoxic, antibacterial
Pterulone (20) and pterulinic acid (21) Pterula sp. Antifungal
Nafuredin (22) Aspergillus niger Anthelmintic
Paecilaminol (33) Paecilomyces sp. Anthelmintic, insecticidal
Verticipyrone (37) Verticillium sp. Anthelmintic, insecticidal
Ukulactones A (52) and B (53) Penicillium sp. Anthelmintic
Complex II inhibitors
Harzianopyridone (58) Trichoderma harzianum Antifungal
Atpenins A4 (59) and A5 (60) Penicillium sp. Antifungal
Siccanin (12) Helminthosporium siccans Antifungal
Anhydrofulvic acid (71) Penicillium spp. Antifungal
Complex III inhibitors
Strobilurin A (7) Strobilurus tenacellus Antifungal
Ilicicolin H (64) Cylindrocladium ilicicola Antifungal, cytotoxic
Funiculosin (65) Penicillium funiculosum Antifungal, antiviral
Sambutoxin (66) Fusarium sambucinum Platelet aggregation inhibitor
Oudemansin A (74) Oudemansiella mucida Antifungal
Complex V inhibitors
Aurovertin B (77) Calcarisporium arbuscula Apoptosis inducer
Citreoviridin (78) Penicillium citreoviride Neurotoxic
Asteltoxin (79) Aspergillus stellatus Toxic
Efrapeptin D (80) Tolypocladium niveum Antimalarial, antifungal, insecticidal
Tentoxin (81) Alternaria tenuis Chloroplast F1-ATPase Inhibitor, phytotoxic
Uncouplers
ACR-toxin I (82) Alternaria citri Phytotoxic
Leucinostatin A (83) Penicillium lilacinum Antifungal, antimalarial

complex I of anaerobic helminths. One com- phosphorylation inhibitors of fungal origin, in-
pound, atpenin A5, inhibited complex II potently. cluding the NFRD inhibitors. These inhibitors
Here, we review electron transport and oxidative are summarized in Table 12.1.
250 Rokuro Masuma et al.
II. Inhibitors of Complex I
Complex I (NADH-ubiquinone reductase) is a rela-
tively massive enzyme. In mammals, complex I con-
sists of 45 different subunits with a combined
molecular mass approaching 1 MDa, together with
noncovalently bound FMN and eight iron–sulfur
clusters (Carroll et al. 2006). Electrons from NADH
are accepted by ubiquinone through complex I,
ubiquinone being reduced to ubiquinonol. In this
process, protons are transferred from the mitochon-
drial matrix to the intermembrane space, thus pro-
ducing the electrochemical proton gradient. This is
the first step of the electron transport system.
All known high-affinity inhibitors of complex
I act at the terminal electron transfer step (qui-
none-binding site). They are classified into two or
three groups (Friedrich et al. 1994; Degli Esposti
and Ghelli 1999; Okun et al. 1999). Type A (class I)
inhibitors, including piericidin A (10) and fenpyr-
oximate (3), are quinone antagonists and inhibit
in a partially-competitive manner with regard to
ubiquinone. Type B (class II) inhibitors, including
rotenone (9), aureothin (Fig. 12.5, 13) and phe-
noxan (Fig. 12.5, 14), are semiquinone antagonists
and inhibit in a noncompetitive fashion (Washizu
et al. 1954; Kunze et al. 1992). Aureothin is pro-
duced by Streptomyces sp. and 14 is produced by
myxobacteria. Type C inhibitors are quinol anta-
gonists, such as capsaicin (Fig. 12.5, 15), a pun-
gent principle of chili peppers (Shimomura et al.
1989; Yagi 1990). However, this classification does
not mean the existence of two or three distinct
inhibitors and quinone-binding sites. Okun et al.
(1999) proposed the existence of only one large
NO2
O CH3
O H3CO
H3CO
H 3C CH3
O
Aureothin (13)
O
H3CO
N
H
HO
Capsaicin (15)
Fig. 12.5. Structures of aureothin, phenoxan, and capsaicin
inhibitor binding pocket in the hydrophobic part
of complex I.
Many natural complex I inhibitors have been
reported, especially from plants and myxobac-
teria, whereas the number of reported fungal com-
plex I inhibitors is very small.
Cochlioquinones (Fig. 12.6) are isolated from
Cochliobolus miyabeanus and some other fungi
(Carruthers et al. 1971). They have a structure of
benzoquinone joined to a sesquiterpene and they
show various biological activities, such as nema-
tocidal (against Caenorhabditis elegans), phyto-
toxic and anti-angiogenic properties and
competitive inhibition of specific [3H]ivermec-
tin-binding (Schaeffer et al. 1990). Inhibitory
activities (IC50 values) of cochlioquinones A (16)
and B (17), isocochlioquinone A (18) and stem-
phone A (19) against bovine heart NADH oxidase
(complexes I+III+IV) were 115, 83, 56 and 160
nmol/mg of protein, respectively (Lim et al.
1996). Compound 17 inhibited complex I at an
IC50 value of 370 nmol/mg of protein. It did not
inhibit the other complexes, which indicated 17
was a specific complex I inhibitor. The inhibition
against NADH is uncompetitive but inhibition
against quinone changes from noncompetitive to
competitive when the exogenous quinone concen-
tration increases. A similar complicated inhibition
against complex I was reported for capsaicin (15),
a type C inhibitor of complex I (Yagi 1990).
Pterulone (Fig. 12.6, 20) and pterulinic acid
(Fig. 12.6, 21) are produced by the submerged
culture of the basidiomycete Pterula sp. (Engler
et al. 1997). They have a 1-benzoxepin ring
with chloromethylidene. Though 20 is a single E
O
O
N
H 3C CH3 CH3
O
Phenoxan (14)
CH3
CH3
 Helminth Electron Transport Inhibitors Produced by Fungi 251

CH3 H3C O CH3 H3C O CH3

R2 O O
CH3 CH3 CH3
O HO O
CH3 CH3 CH3
R1 O HO
O OH O
H3C CH3 CH3 CH3 H3C CH3
O O O
H H H
H 3C H3C H3C
O O O
HO H H HO H H HO H H
CH3 CH3 CH3
R1 R2
Cochlioquinone A (16) OH OCOCH3 Isocochlioquinone A (18) Stemphone A (19)
Cochlioquinone B (17) H =O

O O
H3C Cl HO
CH2 Cl
O O

Pterulone (20) Pterulinic acid (21)

Fig. 12.6. Structures of complex I inhibitors produced by fungi

isomer, 21 is a 5:1 mixture of E:Z that cannot be complex II (rhodoquinol-fumarate reductase).

separated from each other. The compounds inhib-
it the growth of fungi. The IC50 values of 20 and 21
against bovine heart NADH oxidase were 36 and
450 mM, respectively, while they did not inhibit
succinate oxidase (complexes II+III+IV). There-
fore, they may be complex I inhibitors.
Since complex I reduces ubiquinone and com-
plex III oxidizes ubiquinol, both complexes have
quinone-binding sites. Therefore, some inhibitors
targeting quinone-binding sites of complex I in-
hibit complex III and vice versa (Degli Esposti
et al. 1993). Strobilurin A (7) inhibits complex
III potently (see Sect. V.A) and reportedly inhibits
bovine heart complex I weakly (15.5% inhibition
at 3.3 mM; Degli Esposti et al. 1993).
Therefore, the anaerobic complex II catalyzes the
reverse reaction of aerobic complex II (succinate-
ubiquinone reductase). The end-products of this
glucose catabolism are volatile fatty acids, such as
2-methylpentanoate. This anaerobic electron trans-
port system can provide ATP in the absence of
oxygen. Rhodoquinone is used for this anaerobic
respiration. Having a lower redox potential than
ubiquinone enables rhodoquinone to be used for
the reverse reaction of aerobic complex II. NFRD,
composed of the above complex I and complex II,
plays an important role in the anaerobic respiratory
system. Therefore, NFRD inhibitors have been
sought from microbial origin to produce anthel-
mintics and some helminth-specific complex I inhi-
bitors have been discovered.

III. Inhibitors of Helminth Complex I

A. NADH-Fumarate Reductase
Energy metabolism in many adult helminths
differs from that in larvae and host (Kita et al.
2001; Komuniecki and Tielens 2003). They produce
ATP in low oxygen concentration using a special
respiratory system (Fig. 12.7). Phosphoenolpyr-
uvate produced via an anaerobic glycolytic pathway
is converted to oxalacetate by phosphoenolpyr-
uvate carboxykinase, and oxalacetate is metabo-
lized to malate and then to fumarate. Electrons
from NADH are accepted by rhodoquinone
through complex I (NADH-rhodoquinone reduc-
tase) and then transferred to fumarate through
B. Nafuredin
1. Producing Strain and Fermentation
Nafuredin (Fig. 12.8, 22) was isolated as an NFRD
inhibitor (Ōmura et al. 2001; Ui et al. 2001). The
producing fungal strain, FT-0554, was isolated from
a marine sponge collected in the Palau Islands, Re-
public of Palau. The strain FT-0554 was identified as
Aspergillus niger from morphological characteris-
tics. A. niger is well known as a terrestrial fungus, so
the effect of seawater concentration in a culture
medium on fungal growth and nafuredin produc-
tion was studied (Masuma et al. 2001).
The mycelial growth and the production of
22 were evaluated in different natural seawater
252 Rokuro Masuma et al.
Fig. 12.7. Difference of energy
metabolism between aerobic
mammals and anaerobic hel-
minths
CH3 CH3 CH3 CH3
O
CH3
O
HO
O
Nafuredin (22)
Fig. 12.8. Structure of nafuredin
concentrations (0–100%) after incubation at 25
C
for 7 days on potato-dextrose agar or broth. The
mycelial growth of strain FT-0544 increased with
natural seawater concentration, and the addition of
natural seawater (25–100%) enhanced the produc-
tion of nafuredin. The strain FT-0554 also grew
abundantly on medium containing 4% of sodium
chloride. Generally, the growth of terrestrial fungi
tends to be suppressed in the presence of natural
seawater. Therefore, A. niger FT-0554 is suggested
to be adapted to the marine environment.
After the isolation of 22, more than five fungal
strains have been found as producers of 22 during
the screening of NFRD inhibitors. It is interesting
that all strains were terrestrial fungi, and more-
over, all were Trichoderma spp.
2. Structure
The structure of 22 was elucidated by NMR and
mass spectra analysis (Ui et al. 2001). The total
synthesis of 22 revealed its absolute configuration
(Takano et al. 2001a, b). It has a b,g-epoxy-d-
lactone ring with an alkenyl side chain. The bio-
synthesis study by Ui et al. (2001) suggested that
22 is composed of nine acetates and four methio-
nines (branched four methyl carbons).
Many natural d-lactones have been reported.
d-Decalactone (Fig. 12.9, 23) is found in several
kinds of foods and it is also produced by fungi
(Nago et al. 1993). It is used for flavoring and
fragrance. With the exception of the simple lac-
tones, natural alkenyl-d-lactones are usually very
cytotoxic. Leptomycin B (Fig. 12.9, 24) and kazu-
samycin A (Fig. 12.9, 25), produced by Streptomy-
ces spp., are antitumor and antifungal compounds
(Hamamoto et al. 1983; Umezawa et al. 1984).
They inhibit nuclear export signal-dependent nu-
clear export of proteins (Wolff et al. 1997). Fos-
triecin (Fig. 12.9, 26) and pironetin (Fig. 12.9, 27)
are also antitumor compounds produced by Strep-
tomyces spp. (Tunac et al. 1983; Kobayashi et al.
1994). The former inhibits protein phosphatase
2A (Roberge et al. 1994) and the latter causes
microtubule disassembly (Kondoh et al. 1999).
Aurovertins, citreoviridin and asteltoxin are fun-
gal metabolites possessing alkenyl-d-lactones.
They are mycotoxins and inhibit F1-ATPase of
complex V (see Sect. V.B). ACR-toxin I is a fungal
phytotoxin produced by Alternaria spp. It is an
uncoupler (as shown in Sect. V.C).
Natural compounds having a b,g-epoxy-d-lac-
tone moiety have only been found in clerodane
furanoditerpene group, such as palmarin (Fig.
12.9, 28), isolated as a bitter component of
Calumba root (Wessely et al. 1936), but their
d-lactones are parts of fused rings (Yonemitsu
et al. 1989). Therefore, 22 is the first natural com-
pound having nonfused b,g-epoxy-d-lactone.
 Helminth Electron Transport Inhibitors Produced by Fungi 253

CH3
CH3 O OH
CH3 COOH
O O CH3 CH3 R CH3 CH3 CH3

O O
R
d-Decalactone (23) Leptomycin B (24) CH3
Kazusamycin A (25) CH2OH
O

CH3 CH3 CH3 O

H3C OH H H
CH3
O
O O CH3
O H2O3PO OH O OH OCH3
OH
CH3
O O O OH

Fostriecin (26) Pironetin (27) Palmarin (28)

Fig. 12.9. Structures of d-lactones produced by microorganisms

3. Enzyme Inhibition and Biological Activity
The screening for NFRD inhibitors was carried out
using Ascaris suum (roundworm) mitochondria.
Compound 22 inhibited NFRD at an IC50 value of
12 nM (Table 12.2; Ōmura et al. 2001). Since NFRD
consists of NADH-rhodoquinone reductase (com-
plex I) and rhodoquinol-fumarate reductase (com-
plex II), inhibitory activity against each enzyme of
A. suum was evaluated. As shown in Table 12.2, 21
potently inhibited complex I (IC50 = 24 nM) and
the inhibition against complex II was very weak
(IC50 = 80 mM). It is interesting that 22 inhibited
not only anaerobic adult complex I of A. suum
(NADH-rhodoquinone reductase) but also larval
complex I (NADH-ubiquinone reductase), which
has an aerobic energy metabolism like that of
mammals. The IC50 value against larval complex I
was 8.9 nM. Therefore, 22 is a complex I inhibitor.
However, 22 only inhibited rat liver complex I at
very high concentration (IC50 = 10 mM). Therefore,
22 seems to be a selective inhibitor of helminth
complex I. From a kinetic study against NADH-
rhodoquinone reductase of adult A. suum, 22
revealed competitive inhibition with rhodoqui-
none and noncompetitive inhibition with NADH,
which indicated that the site of inhibition of 22 is
the quinone-binding domain in complex I.
Haemonchus contortus (barberpole worm) is
reported to have an NFRD system (Van Helle-
mond et al. 1995). Therefore, in vivo anthelmintic
activity of 22 was evaluated using H. contortus-
infected sheep (Ōmura et al. 2001). As shown in
Table 12.3, 22 (2 mg/kg p.o.) significantly reduced
the number of fecal eggs of infected sheep. This
anthelmintic activity may be due to the inhibition
of complex I, because 22 also inhibits the enzyme
of H. contortus (Table 12.2). Moreover, 22 was
effective in mice infected with dwarf tapeworm,
Hymenolepis nana. There were no signs of any
side-effects and no loss of body weight during
tests in either sheep (2 mg/kg p.o.) or mice (50
mg/kg p.o. and i.p.).

Table 12.2. Effects of nafuredin on electron transport enzymes of nematodes and rat. NT Not tested

Enzyme Complex IC50 (nM)

Ascaris suum Ascaris Haemonchus Rat liver
(adult) suum (L2) contortus (adult)
NADH-fumarate reductase I+II 12 NT NT 1000
NADH-ubiquinone reductase I 8 8.9 86 10 000
NADH-rhodoquinone reductase I 24 9.0 195 >100 000
Rhodoquinol-fumarate reductase II 80 000 NT NT NT
Succinate-ubiquinone reductase II >100 000 NT NT >100 000
254 Rokuro Masuma et al.

Table 12.3. Effects of treatment with nafuredin on fecal some analogs (Fig. 12.11). The inhibitory activities
egg counts in sheep infected with Haemonchus contortus. of the 2-deoxy derivative (30) and C4 epimer (31)
Nafuredin (2 mg/kg) was given orally at day 0. Values are
means of three experiments (treated animals) or two
against NFRD of adult A. suum were similar to
experiments (controls)  SD that of 29. Therefore, the enol (or 2-ketone) func-
tionality and the C4 stereochemistry of 29 are not
Number of eggs per gram of feces responsible for the inhibitory activity. However,
Treated Control the NFRD inhibitory activity of the C5 epimer (32)
Day 1 5689  1120 5200  754 was weakened 20-fold, which suggests that
Day 4 1489  655 5100  424 the stereochemistry of the C5 hydroxy group is
Day 11 289  214 4034  801 important for the inhibitory activity of 29.

4. Nafuredin-g and its Analogs

During total synthesis of 22, it was found to convert
into a novel g-lactone derivative, named nafuredin-
g (Fig. 12.10, 29), under mild basic conditions
(Nagamitsu et al. 2003; Shiomi et al. 2005). The
epoxide of 22 opens and the d-lactone recyclizes
to form g-lactone with keto–enol tautomerism.
Compound 29 inhibited NADH-rhodoquinone re-
ductase of adult A. suum with an IC50 value of 2.3
nM, and did not inhibit bovine liver NADH-ubi-
quinone reductase at 10 mM. It showed anthelmin-
tic activity against H. contortus using treatment
with two oral doses each of 2 mg/kg. Thus, 29 has
similar enzyme inhibitory and anthelmintic activ-
ities as those of 22. Though 22 was converted
into 29 in basic condition, only a part of 22 was
converted in neutral buffer. Therefore, it is not
likely that 21 was converted into 29 and showed
inhibitory activity. Both 22 and 29 may inhibit
complex I directly.
Since the lactone moiety synthesis of 29 is
simpler than that of 22, 29 is useful for studying
structure-activity relationships. The total synthe-
sis of 29 has been achieved (Nagamitsu et al.
2003), and Nagamitsu et al. (2008) prepared
C. Paecilaminol
Paecilaminol (Fig. 12.12, 33) was produced by a
fungus, Paecilomyces sp. FKI-0550, isolated from a
soil sample collected on Miyakojima Island,
Okinawa Prefecture, Japan (Ui et al. 2006a). The
structure of 33 was elucidated by NMR and mass
spectra as 2-amino-14,16-dimethyl-3-octadeca-
nol. Compound 32 inhibited the growth of
free-living nematode C. elegans and brine shrimp
Artemia salina at the MIC values of 20 mg/ml and
5 mg/ml, respectively.
The adult Ascaris suum NFRD inhibitory
activity of 33 was moderate (IC50 = 5.1 mM), but
the inhibition was about four times more potent
than that of bovine heart NADH oxidase
(complexes I+III+IV, IC50 = 19.8 mM). The IC50
values of 33 against NADH-rhodoquinone reduc-
tase (complex I) and rhodoquinol-fumarate reduc-
tase (complex II) of A. suum were 23 mM and 35 mM,
respectively. The values against NADH-ubiquinone
reductase (complex I) and ubiquinol–cytochrome-c
reductase (complex III) of bovine heart were 16 mM
and 20 mM, respectively. However, 33 does not
inhibit bovine succinate-ubiquinone reductase

CH3 CH3 CH3 CH3 CH3 – CH3

O O O
CH3 K2CO3
O O HO O
HO MeOH, r.t. HO
H
O O O
–
Base
Naf uredin (22)

HO O
CH3 CH3 CH3 CH3 CH3 CH3 CH3
O CH3 O
CH3 CH3
O O
OH OH
Naf uredin - g (29)

Fig. 12.10. Proposed mechanism of the conversion of nafuredin to nafuredin-g

 Helminth Electron Transport Inhibitors Produced by Fungi 255

HO
3 CH3 CH3 CH3 CH3 CH3 CH3
O CH3 O CH3
1 CH3 CH3
O 5 6 10 16 O
OH OH
Nafuredin-g (29) 2-Deoxynafuredin-g (30)
IC50 = 6 nM IC50 = 8 nM

HO HO
CH3 CH3 CH3 CH3 CH3 CH3
O CH3 O CH3
O CH3 O CH3

OH OH
(4S)-Nafuredin-g (31) (5S)-Nafuredin-g (32)
IC50 = 7 nM IC50 = 120 nM

Fig. 12.11. NADH-fumarate reductase inhibitory activities of nafuredin-g analogs

OH OH

H3C CH3 H3C OH

CH3 CH3 NH2

NH2

Paecilaminol (33) Sphingosine (34)

HOOC O
HOOC O OH OH
CH3 H3C CH3
H3C
CH3 CH3 OH NH2 OH
HOOC O

HOOC O

Fumonisin B1 (35) 2-Decanol (36)

Fig. 12.12. Structures of paecilaminol and its related compounds

(complex II) at 100 mM. Therefore, 33 shows similar
inhibitory activities against complexes I, II and III
of A. suum and bovine heart, except bovine com-
plex II. It is not common for electron transport
inhibitors to show such low selectivity. The only
group that show such wide inhibitions are 2-alkyl-
4,6-dinitrophenols, with their inhibitory activities
against complex II also being weaker than that for
complexes I and III (Tan et al. 1993). The low
selectivity of 33 may be due to its linear structure,
because both amino and hydroxy groups can freely
rotate and be attached to enzymes.
Compound 33 is an amino alcohol. The NFRD
inhibitory activity of a similar amino alcohol
sphingosine (Fig. 12.12, 34), a long-chain base of
sphingolipids, was weaker (IC50 = 28 mM) than 33.
However, Fumonisin B1 (Fig. 12.12, 35), an amino
alcohol produced by the fungus Fusarium monili-
forme (Gelderblom et al. 1988), did not inhibit
NFRD at 100 mM. Desai et al. (2002) reported
that fumonisins inhibited ceramide synthase
(sphingosine N-acyltransferase). A simple alcohol
2-decanol (Fig. 12.12, 36) also showed no inhibi-
tion against NFRD at 100 mM.
D. Verticipyrone
Verticipyrone (Fig. 12.13, 37) was produced by a
fungus, Verticillium sp. FKI-1083, isolated from
a soil sample collected on Yakushima Island,
Kagoshima Prefecture, Japan (Ui et al. 2006b).
The structure was shown to be (E)-2-
methoxy-3, 5-dimethyl-6-(3-methyl-2-undecenyl)
256 Rokuro Masuma et al.

CH3 CH3
H3CO O CH3 H3CO O
CH3
H3C CH3 H3C CH3
O O

Verticipyrone (37): IC50 = 4.1 nM 38: IC50 = 2.0 nM

CH3
H3CO O CH3
H3CO O CH3
H3C CH3
H3C CH3
O
O

39: IC50 = 4.7 nM 40: IC50 = 4.8 nM

CH3
H3CO O CH3
H3CO O CH3
H3C OH
H3C OH CH3
CH3
O
O

41: IC50 = 0.65 nM 42: IC50 = 0.30 nM

CH3
H3CO O CH3
H3CO O CH3

OH H3C CH3
O
O

43: IC50 > 100 nM 44: IC50 > 100 nM

Fig. 12.13. NADH-fumarate reductase inhibitory activities of verticipyrone and its analogs

-4H-pyran-4-one by NMR and mass spectra
studies. MIC values of 37 against Caenorhabditis
elegans and A. salina were 20 mg/ml and 2.0 mg/
ml, respectively.
Compound 37 inhibited NFRD from A. suum
with an IC50 value of 4.1 nM and the inhibition
was specific to NADH-rhodoquinone reductase
(complex I) as shown in Table 12.4. However, its
inhibitory activity against NADH-ubiquinone re-
ductase (complex I) from bovine heart was similar
to that of A. suum complex I.
The total synthesis of 37 has been accom-
plished and some analogs of 37 were prepared
(Shimamura et al. 2007), as shown in Fig. 12.13.
Olefin isomers 38 and 39 and alkyl side chain
analog 40 showed Ascaris NFRD inhibitory activ-
ities similar to 37, which suggests that the olefin in
the side chain is not important. The two alcohols
41 and 42 showed much potent NFRD inhibitory
activities than 37 suggesting the newly introduced
hydroxy group on the side chain may contribute
to the inhibition. However, 43 (didemethyl 41)
showed no NFRD inhibition. Therefore, 3,5-di-
methyl groups on the g-pyrone moiety may be
essential for NFRD inhibition. The g-pyrone 44
showed no NFRD inhibition, which suggests
that the long side chain is also important for the
inhibition.
Among the analogs, 41 showed >20-fold more
potent inhibition against Ascaris complex I com-
pared with that of 37, whereas inhibition of 41
against bovine heart complex I was four times
less potent than that of 37 (Table 12.4). Therefore
41 is a selective inhibitor of Ascaris complex I.
Alveolar echinococcosis caused by larval Echi-
nococcus multilocularis is a life-threatening parasit-
ic zoonosis. Recently, E. multilocularis was found to
use an anaerobic NADH-fumarate reductase system
for its energy metabolic pathways (Matsumoto et al.
2008). Therefore, the efficacy of 42 (the most potent
NFRD inhibitor among the analogs of 37) against
larval E. multilocularis was evaluated. The viability
of the E. multilocularis protoscolex was progres-
sively reduced during in vitro treatment of the
 Helminth Electron Transport Inhibitors Produced by Fungi
Table 12.4. Effects of verticipyrone and its analog 41 on electron transport enzymes
Enzyme
Ascaris suum NADH-fumarate reductase
NADH-rhodoquinone reductase
Rhodoquinol-fumarate reductase
Bovine heart NADH oxidase
NADH-ubiquinone reductase
Succinate-ubiquinone reductase
Ubiquinol–cytochrome-c reductase
parasites with 42, and more than 90% of the para-
sites were eliminated on days 5 and 18 when 42 was
used at 50 mM and 5 mM, respectively (Matsumoto
et al., unpublished data).
Compound 37 has the structure of 2-methoxy-
3,5-dimethyl-g-pyrone with a side chain at C6.
Aureothin (13) produced by Streptomyces thio-
luteus has the same skeleton and inhibits complex
I as shown in Sect. II. Neoaureothin (spectinabilin;
Fig. 12.14, 45), an analog of 13 produced by Strep-
tomyces spp. (Cassinelli et al. 1966), was found to
inhibit Ascaris NFRD with an IC50 value of 15 nM
(Ui et al. 2006b).
Some g-pyrones with a side chain at C6 have
been also isolated from the culture broths of fungi.
Himeic acid A (Fig. 12.14, 46), produced by Asper-
gillus sp. (Tsukamoto et al. 2005) inhibited
ubiquitin-activating enzyme (E1). Carbonarone
A (Fig. 12.14, 47) produced by Aspergillus carbo-
narius (Zhang et al. 2007) showed moderate cyto-
toxicity. The producing strains of 46 and 47
were both marine-derived fungi. Funicone (48),
deoxyfunicone (49) and vermistatin (50) have
(E)-propenyl side chains at C6 of the g-pyrone
(Fig. 12.14). They were produced by Penicillium
funiculosum, P. vermiculatum and some other
fungi (Merlini et al. 1970; Fuska et al. 1979; Sassa
et al. 1991). Compounds 48 and 49 showed anti-
fungal activities and 50 showed cytotoxicity. Com-
pounds 49 and 50 also potentiated the antifungal
activity of miconazole against Candida albicans
(Arai et al. 2002). 2,3,5-Trimethyl-6-(3-oxobutan-
2-yl)-4H-pyran-4-one (Fig. 12.14, 51), produced
by Aspergillus sydowi isolated from the deep sea,
was not cytotoxic (Li et al. 2007).
E. Ukulactones
Ukulactones A and B (Fig. 12.15, 52 and 53) were
produced by a fungus, Penicillium sp. FKI-3389,
isolated from a soil sample collected on Hawaii
257
Complex IC50 (nM)
Verticipyrone 41
I+II 4.1 0.65
I 49 2.0
II >100 000 >100 000
I+III+IV 1.3 20
I 46 200
II >100 000 >100 000
III 26 000 80 000
Island, Hawaii, United States (Ōmura et al. 2007).
They have the same planar structure, with 2-oxa-
bicyclo[2.2.1]heptane-3,5-dione and 5,6-dihydro-
2H-pyran linked by pentaene. The configurations
at C2 of the pyran (junction of the pyran and the
pentaene) are opposite between 52 and 53. The
absolute configurations of 52 and 53 have not yet
been elucidated. Compound 52 inhibited the
growth of parasite nematode Nippostrongylus bra-
siliensis at 1 mg/ml in vitro.
Compound 52 inhibited Ascaris NFRD (Table
12.5) and is an NADH-rhodoquinone reductase
(complex I)-specific inhibitor (IC50 = 55 nM). Its
inhibition against bovine heart NADH-ubiqui-
none reductase (complex I) was about 500 times
weaker (IC50 = 28 mM) than that of Ascaris com-
plex I. The inhibitions against bovine complexes
II and III were also weak. Therefore, 52 is a
selective inhibitor of Ascaris complex I, compa-
rable to nafuredin (22). The difference between
52 and 53 is the configuration of only one carbon.
However, the inhibition of 53 against Ascaris
NFRD was about 200 times weaker than that of
52 (Table 12.5). Thus, the configuration at C2 of
the pyran may be very important for the inhibi-
tory activity.
Recently, demethyl 52 compound, prugosene
A1 (Fig. 12.15, 54), was isolated from the surface-
cultured mycelium of a marine sponge-derived
Penicillium rugulosum by Lang et al. (2007). Com-
pound 54 had no antimicrobial activity. Dong et al.
isolated 195-A (Fig. 12.15, 55) from the culture
broth of Talaromyces wortmannii. The pentaene
moiety of 54 is altered to tetraene at 55. 2-Oxabicy-
clo[2.2.1]heptane-3,5-dione is also found in
the structure of shimalactone A (Fig. 12.15, 56)
produced by the marine fungus Emericella varieco-
lor (Wei et al. 2005). Compound 56 induced neur-
itogenesis against neuroblastoma cells. Compounds
52 and 53 have an (all-E)-2,10-dimethyldodeca-2,
4,6,8,10-pentaene moiety, which is also found in
258 Rokuro Masuma et al.

NO2 NO2
O CH3 O CH3 CH3 CH3
H3CO O H3CO O

H 3C CH3 H3C CH3

O O
Aureothin (13) Neoaureothin (Spectinabilin, 45)

O COOH O
H
HO2C N H2N

CH3 O O O O O

Himeic acid A (46) Carbonarone A (47)

OCH3 O CH3
H3CO O CH3 OCH3

R
O O O O
H3CO O H3CO
R O
Funicone (48) OH Vermistatin (50)
Deoxyf unicone (49) H

CH3
H3C O CH3

H3C O
CH3
O
2,3,5-Trimethyl-6-(3-oxobutan-2-yl)-4H-pyran-4-one (51)

Fig. 12.14. Structures of verticipyrone related g-pyrones

phenalamide A2 (57) produced by the myxobacter-
ium Myxococcus stipitatus (Trowitzsch-Kienast
et al. 1992). It suppressed HIV-1 replication in cell
cultures and inhibited NADH-ubiquinone reduc-
tase (complex I) potently (Friedrich et al. 1994).
It is interesting that mammalian complex I was
inhibited potently by 57, but not by 52 and 53.
IV. Inhibitors of Complex II
A. Atpenins and Harzianopyridone
1. Structures
Harzianopyridone (Fig. 12.16, 58), produced by
the fungus Trichoderma sp. was isolated during
screening for NFRD inhibitors (Miyadera et al.
2003). It was originally isolated from T. harzia-
num and showed antifungal, antibacterial and
herbicidal activities, but its mode of action had
not been studied (Dickinson et al.1989; Cutler and
Jacyno 1991). Structurally similar antifungal anti-
biotics, atpenins A4, A5 and B (Fig. 12.16, 59–61),
have been isolated from the culture broth of the
fungus Penicillium sp. (Ōmura et al. 1988; Kuma-
gai et al. 1990) before the report of 58. Since 61
was suggested to inhibit the ATP-generating sys-
tem (Oshino et al. 1990), the effects of 58 together
with atpenins on the inhibitory activities of elec-
tron transport enzymes were examined and were
revealed to be potent and selective complex II
(succinate-ubiquinone reductase) inhibitors, as
shown below.
Though the structure of 58 is depicted as 2-
pyridone and the structures of 59-61 are depicted
as 2-pyridinol in Fig. 12.16, structures in the figure
only reflect those of the original papers. 2-Pyri-
done and 2-pyridinol are tautomers, and they may
be equivalent. The other compounds having the
same chromophore are WF-16775 A1 and A2
(Fig. 12.16, 62, 63), isolated from the fungus
 Helminth Electron Transport Inhibitors Produced by Fungi 259

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

O CH3 CH3
O
HO O HO O
O O
H3C O O
H3C H3C
CH3 H 3C
CH3

Ukulactone A (52) Ukulactone B (53)

CH3 CH3 CH3 CH3 CH3 CH3

O CH3 CH3
O
HO O HO O
O O
H3C O O
H3C H3C
CH3 H3C
CH3

Prugosene A1 (54) 195-A (55)

H3C CH3
H3C O
CH3 O
H3C H HC O
3
CH3
H CH3 CH3 CH3 CH3 CH3
H CH3
H3C HO H N
HO
CH3 CH3 O OH

Shimalactone A (56) Phenalamide A2 (57)

Fig. 12.15. Structures of ukulactones and their related compounds

Table 12.5. Effects of ukulactones on electron transport enzymes. NT Not tested

Enzyme Complex IC50 (nM)

Ukulactone A Ukulactone B
Ascaris suum NADH-fumarate reductase I+II 2.4 470
NADH-rhodoquinone reductase I 55 NT
Rhodoquinol-fumarate reductase II >100 000 NT
Bovine heart NADH oxidase I+III+IV 9000 16 000
Succinate–cytochrome-c reductase II+III 68 000 30 000
NADH-ubiquinone reductase I 28 000 NT
Succinate-ubiquinone reductase II >100 000 NT
Ubiquinol–cytochrome-c reductase III 32 000 NT

Chaetasbolisia erysiophoides (Otsuka et al. 1992),
and they display potent angiogenetic activity.
Fungi also produce some 2-pyridone com-
pounds. Ilicicolin H (Fig. 12.17, 64) was isolated
by Hayakawa et al. (1971). Funiculosin
(Fig. 12.17, 65) and sambutoxin (Fig. 12.17, 66)
are N-methyl-2-pyridones (Ando et al.1969;
Kim et al. 1995). Compounds 64–66 inhibit
complex III (as shown in Sect. V.A). Flavipucine
(Fig. 12.17, 67), produced by Aspergillus flavipes
(Findlay and Radics 1972), shows antibacterial
and fungicidal activities, and apiosporamide
(Fig. 12.17, 68), produced by Apiospora montag-
nei (Alfatafta et al. 1994), possesses antifungal
activity. Militarinone D (Fig. 12.17, 69), isolated
from the culture broth of Paecilomyces militaris
(Schmidt et al. 2003), exhibits cytotoxicity.
260 Rokuro Masuma et al.

OH O OH O Cl OH O Cl
H3CO CH3 H3CO H3CO Cl
CH3
H3CO N O CH3 H3CO CH CH3
OH 3 H3CO N CH CH3
OH 3
H N

Harzianopyridone (58) Atpenin A4 (59) Atpenin A5 (60)

OH O OH O Cl OH O Cl
H3CO CH3 H3CO Cl H3CO Cl
Cl
H3CO CH CH3 CH CH3 H3CO CH CH3
N OH 3 H3CO N OH 3 N OH 3

Atpenin B (61) WF-16775 A1 (62) WF-16775 A2 (63)

Fig. 12.16. Structures of atpenins and their related compounds

Fig. 12.17. Structures of 2-pyridones produced by microorganisms

As for Actinomycetes, most 2-pyridone-type an antibacterial antibiotic and inhibits protein

products belong to the kirromycin (Fig. 12.17, 70) synthesis interfering with transpeptidation by act-
group. Kirromycin (mocimycin), produced by ing on elongation factor Tu (Parmeggiani and
Streptomyces collinus (Wolf and Zähner 1972), is Nissen 2006).
 Helminth Electron Transport Inhibitors Produced by Fungi
2. Enzyme Inhibition and Biological Activity
The effects of 58–60 on electron transport enzymes
are shown in Table 12.6 (Miyadera et al. 2003).
Though they inhibited NFRD as potently as nafur-
edin (22), verticipyrone (37) and ukulactone A (52),
they did not inhibit complex I. However, they inhib-
ited Ascaris rhodoquinol-fumarate reductase and
bovine heart succinate-ubiquinone reductase.
Therefore, they are specific complex II inhibitors
and not just selective for helminths. The inhibition
against mammalian complex II is more potent than
that of helminth complex II. Atpenins inhibited
complex II more potently than 58, and 60 exhibited
the most potent inhibition. Carboxin (1) is known
as a potent complex II inhibitor (Mowery et al.
1977). However, the IC50 value against bovine
heart succinate-ubiquinone reductase was 1.1 mM,
which is 300 times weaker than that for 60. There-
fore, atpenins may be useful tools for clarifying the
biochemical and structural properties of complex II
(Martens et al. 2005; Adebiyi et al. 2008).
X-ray crystallographic analyses of Escherichia
coli and porcine heart complex II have been
reported (Yankovskaya et al. 2003; Sun et al.
2005). Each complex is composed of four subu-
nits, FAD-containing flavoprotein (Fp or SdhA),
iron-sulfur protein (Ip or SdhB) and two mem-
brane anchor subunits, CybL (SdhC) and CybS
(SdhD). The succinate dehydrogenase catalytic
portion is formed by Fp and Ip. The membrane
anchor subunits are required for electron transfer
to ubiquinone. Ubiquinone binds to complex II
at the interface between Ip and the membrane
anchor subunits. Kinetic analyses of atpenins
revealed that they exhibited mixed inhibition
with ubiquinone (Ki = 1.0 nM, Ki0 = 5.9 nM
for 60; Miyadera et al. 2003). This indicated that
atpenins may block electron transfer between
the enzyme and ubiquinone by binding to a region
that partly overlaps with the physiological
Table 12.6. Effects of harzianopyridone and atpenins on electron transport enzymes
Enzyme
Ascaris suum NADH-fumarate reductase
NADH-quinone reductase
Rhodoquinol-fumarate reductase
Bovine heart NADH–cytochrome-c reductase
Succinate-ubiquinone reductase
Succinate dehydrogenase
261
ubiquinone-binding site (Q-site). This can explain
the observation that atpenins also affected the
succinate dehydrogenase activity of bovine heart
complex II (Table 12.6).
Compound 60 was co-crystallized with E. coli
succinate-ubiquinone reductase and analyzed in
detail by X-ray crystallography (Horsefield et al.
2006). Yankovskaya et al. (2003) observed that the
interaction between ubiquinone at the Q-site
appeared to be mediated solely by hydrogen
bonding between the carbonyl oxygen (O1) of
ubiquinone and the hydroxy group of tyrosine
83 in SdhD (Q1-site). The co-crystallization study
of complex II and 60 showed that 60 existed in the
same hydrophobic pocket as ubiquinone but
deeper within the pocket (Q2-site). The protein–
ligand docking model of complex II and ubiqui-
none was analyzed in silico, and it revealed that
ubiquinone docked at the Q2-site of complex II.
At the Q2-site, ubiquinone can interact with com-
plex II via additional hydrogen bonds between
carbonyl oxygen (O4) and the hydroxy group of
serine 27 in SdhC and between 4-methoxy group
and the imidazole of histidine 207 in SdhB. These
interactions were observed in the co-crystalliza-
tion result of complex II and 60. The above results
support the proposition that the Q1-site may be
the initial binding site and the Q2-site may be the
catalytic site.
B. Other Complex II Inhibitors
Only a few complex II inhibitors have been found
in microbial metabolites. Siccanin (Fig. 12.18, 12),
produced by the fungus Helminthosporium
siccans, showed antifungal activity, and it is used
clinically for dermatophytosis as an ointment (Ishi-
bashi 1962; Ishibashi et al. 1970). The structure can
be regarded as derived from a cis-fused drimane
condensed with orcinol. It showed 66% inhibition
Complex IC50 (nM)
Harzianopyridone Atpenin A4 Atpenin A5
I + II 1600 110 14
I >100 000 >100 000 >100 000
II 360 220 12
I + III 420 000 140 000 82 000
II 17 11 3.6
II 80 9.2 5.5
262 Rokuro Masuma et al.

OH O HOOC O
CH3
HO
CH3 O
H H
H3C O HO O CH3
CH3

Siccanin (12) Anhydrofulvic acid (71)

O
N CH3

OH

2-n-Heptyl-4-hydroxyquinoline N-oxide (72)

Fig. 12.18. Structures of complex II inhibitors produced by microorganisms

against the succinate dehydrogenase of a fungus,
Trichophyton mentagrophytes, at 0.09 mM (Ishiba-
shi et al. 1970; Nose and Endo 1971). Siccanin is a
species-selective succinate dehydrogenase inhibi-
tor, and it was effective against succinate dehydro-
genases of P. aeruginosa, P. putida, rat and mouse
mitochondria but ineffective or less effective against
those of E. coli, Corynebacterium glutamicum and
porcine mitochondria (Mogi et al. 2009). Anhydro-
fulvic acid (Fig. 12.18, 71) was produced by Penicil-
lium spp. and showed antifungal activity (Wrigley
et al. 1994; Fujita et al. 1999). It exhibited 67%
inhibition against succinate oxidase (complexes II
+III+IV) of a fungus, Candida utilis, at 1.3 mM, but
inhibited NADH oxidase (complexes I+III+IV) only
weakly (18%) at 86 mM, which suggests 71 may be a
complex II inhibitor (Fujita et al. 1999).
As for bacteria, 2-n-heptyl-4-hydroxyquinoline
N-oxide (HQNO; Fig. 12.18, 72) was isolated from
the culture broth of Pseudomonas aeruginosa
(Hays et al. 1945) and reported to inhibit bacterial
complex II. It inhibited succinate-menaquinone
reductase from Bacillus subtilis at the Ki value of
0.2 mM (Smirnova et al. 1995). Compound 72 is well
known as a complex III inhibitor (Izzo et al. 1978).
V. Other Electron Transport Inhibitors
A. Inhibitors of Complex III
Complex III (ubiquinol–cytochrome-c reductase,
cytochrome bc1 complex) accepts electrons from
ubiquinol and passes them to cytochrome c,
thereby transferring protons from mitochondrial
matrix to intermembrane space. Mammalian
complex III is a dimer with a molecular mass
of 490 kDa, and each monomer consists of 11
subunits containing cytochrome b, cytochrome
c1 and an iron–sulfur protein. Cytochrome b has
two quinone-binding sites, an ubiquinol oxidation
site, Qo site (QP site), and a ubiquinone reduction
site, Qi site (QN site). Most complex III inhibitors
bind to either site.
The most famous Qi site inhibitor is antimycin
A3a (11) produced by Streptomyces spp. As for
fungal complex III inhibitors, funiculosin (Fig.
12.19, 65) produced by Penicillium funiculosum
(Ando et al. 1969) and ilicicolin H (Fig. 12.19, 64)
produced by Cylindrocladium ilicicola (Hayakawa
et al. 1971) are Qi site inhibitors (Rotsaert et al.
2008) and possess antifungal activities. While 65
inhibits both yeast and bovine heart complex III at
IC50  10 nM (Rotsaert et al. 2008), the IC50 values
of 64 against yeast and bovine heart complex III
were 3–5 nM and 200–250 nM, respectively (Gutier-
rez-Cirlos et al. 2004). These results suggest a high
degree of specificity in the determinants of ligand-
binding at the Qi site. Sambutoxin (Fig. 12.19, 66) is
structurally related to 64 and 65 and produced by
Fusarium sambucinum (Kim et al. 1995). It was also
reported to inhibit complex III (Kawai et al. 1997)
and may be a Qi site inhibitor. As shown in Sect. IV.
B, HQNO (72) inhibits both complexes II and III. It
is a Qi site inhibitor, but the Kd against bovine heart
mitochondria was about 3 orders of magnitude
 Helminth Electron Transport Inhibitors Produced by Fungi 263

higher than that of antimycin (von Jagow and Link
1986).
The common Qo site inhibitor, myxothiazol
(Fig. 12.19, 73), is produced by the myxobacterium
Myxococcus fulvus (Gerth et al. 1980). It has an E-
b-methoxyacrylamide moiety. Similar E-b-meth-
oxyacrylate moieties are found in some fungal
metabolites, such as strobilurin A (mucidin, 7),
and oudemansin A (Fig. 12.19, 74) produced by
basidiomycetes. Compound 7 was originally iso-
lated from culture broths of Oudemansiella
mucida (Musilek et al. 1969) and Strobilurus tena-
cellus (Anke et al. 1977) and 74 from Oudeman-
siella mucida (Anke et al. 1979). All proved to be
antifungal antibiotics. Their production and prop-
erties have been reviewed by Anke and Erkel
(2002) in The Mycota, Vol. X. Compounds 7 and
74 inhibited bovine heart complex III at IC50
values of 65 nM and 290 nM, respectively (Brandt
et al. 1988), and their targets are also Qo site (von
Jagow and Link 1986). While 7 inhibits both mam-
malian and fungal complex III, its toxicity against
mammals is very weak. Therefore, many analogs
of 7 have been synthesized, and some of them,
such as azoxystrobin (2), are commercially used
for crop protection against phytopathogenic fungi
(Sauter et al. 1999). Compounds having the same
a-substituted methyl (E)-b-methoxyacrylate moie-
ty as 7 and 74 were isolated from the myxobacteria
Cystobacter armeniaca and Archangium gephyra in
2003 (Sasse et al. 2003). They are named cyrmenins,
and cyrmenin B1 (Fig. 12.19, 75) was shown to
inhibit complex III.
As described in Sect. II, some inhibitors target-
ing quinone-binding sites of complex I inhibit
complex III. Compound 7 inhibited bovine heart
complex I weakly (15.5% inhibition at 3.3 mM)
although the inhibition of 73 was more potent
(84.0% inhibition at 3.0 mM). However, 2.9 mM of
65 showed no inhibition against complex I, and Qo
site inhibitors may affect complex I more potently
than Qi site inhibitors (Degli Esposti et al. 1993).
B. Inhibitors of Complex V
The electrochemical proton gradients produced
by the electron transport chain drive complex V

CH3
HO
HO OH CH3
OH O OH
H HO
CH3 CH3
O
HO CH3 CH3 CH3
N O N O
H H
CH3 CH3
Ilicicolin H (64) Funiculosin (65)

HO CH3
OH
CH3 O OCH3 CH3
O S
CH3 CH3 CH3 N CH3
N O H2N
H3C N
CH3 OCH3 S CH3

Sambutoxin (66) Myxothiazol A (73)

CH3
OCH3 H
CH3 N CH2
CH3
O OCH3 O
O OCH3 O NH
H3 C O OCH3
OCH3
OCH3
OCH3

Strobilurin A (7) Oudemansin A (74) Cyrmenin B1 (75)

Fig. 12.19. Structures of complex III inhibitors produced by microorganisms

264 Rokuro Masuma et al.

(ATP synthase, FOF1-ATPase) to produce ATP in
the critical process of oxidative phosphorylation.
Mammalian complex V is suggested to be a dimeric
protein, with each 600-kDa monomer consisting of
15 different protein subunits (Wittig and Schägger
2008). The monomer can be separated into the FO
domain and F1 domain. The FO domain is inserted
in the membrane and translocates protons, while
the F1 domain protrudes into the matrix and
synthesizes ATP. The F1 domain is also called F1-
ATPase because it can hydrolyze ATP to ADP as
the reverse reaction of ATP synthesis.
The name of FO is derived from the oligomy-
cin-sensitive factor (Racker 1963). Oligomycin A
(Fig. 12.20, 76), produced by Streptomyces diasta-
tochromogenes, inhibits proton transport through
the FO domain and is suggested to bind subunits a
and c of the FO domain (Devenish et al. 2000).

Fig. 12.20. Structures of complex V inhibitors produced by microorganisms

 Helminth Electron Transport Inhibitors Produced by Fungi
The subunit composition of the F1 domain is
a3b3g1d1e1, and X-ray analysis of the bovine heart
mitochondrial F1 domain revealed its structure
(Abrahams et al. 1994). Three b-subunits are cat-
alytic. An ADP binds to the first subunit (bDP
subunit), and an ATP binds to the second subunit
(bTP subunit). No nucleotide binds to the third
subunit (bE subunit), and the three catalytic sub-
units interconvert through the cycle of conforma-
tions.
As for F1-ATPase inhibitors, at least five in-
hibitory sites have been identified: nonhydrolysa-
ble NTP analog-binding site (catalytic site),
aurovertin B-binding site, efrapeptin-binding
site, natural inhibitor protein IF1-binding site and
rhodamine 6G-binding site (Gledhill and Walker
2005). A mycotoxin, aurovertin B (Fig. 12.20, 77),
has been isolated from culture mycelia of the
ascomycete Calcarisporium arbuscula (Osselton
et al. 1974) and the basidiomycete Albatrellus con-
fluens (Wang et al. 2005). The KD value of 77
against bovine heart-soluble ATPase was 0.10
mM (Linnett and Beechey 1979). The co-crystalli-
zation study of F1-ATPase and 77 revealed that 77
binds to bovine F1 at two equivalent sites in the
bTP and bE subunits in a cleft between the nucleo-
tide binding and C-terminal domains (van Raaij
et al.1996).
Compound 77 has a 4-methoxy-5-methyl-2-
pyrone with a triene side chain at C-6. The same
moiety is found in two other mycotoxins: citreo-
viridin (Fig. 12.20, 78), produced by the fungus
Penicillium citreoviride (Sakabe et al. 1964), and
asteltoxin (Fig. 12.20, 79), produced by the fungus
Aspergillus stellatus (Kruger et al. 1979). The KD
value of 78 against bovine heart soluble ATPase
was 3.1 mM (Linnett and Beechey 1979). The IC50
value of 79 against rat liver F1-ATPase was about
0.5 mM (Kawai et al. 1997).
Efrapeptin D (Fig. 12.20, 80) is produced by
the fungus Tolypocladium inflatum (Jackson et al.
1979). Efrapeptins are a-aminoisobutyric acid (a-
Aib)-rich peptides with acetylated N-terminus
(Gupta et al. 1992). They are similar to fungal
peptaibols (Whitmore and Wallace 2004) but
their C-terminus is different. Instead of the
amino alcohol C-terminus of peptaibols, efrapep-
tins have pyrrolo[1,2-a]pyrimidine moiety at the
C-terminus. Efrapeptins are mycotoxins and their
antifungal, insecticidal and antimalarial activities
have been reported (Krasnoff et al. 1991; Nagaraj
et al. 2001). Efrapeptins are potent F1-ATPase
265
inhibitors. The KD value of efrapeptins (major
components were 80 and efrapeptins E, F and G)
against bovine heart ATPase was 0.014 mM (Cross
and Kohlbrenner 1978). The co-crystallization
study of F1-ATPase and efrapeptins revealed that
efrapeptins made hydrophobic contact with the a-
helical structure in the g-subunit, which traversed
the cavity, and with the bE subunit and the two
adjacent a subunits (Abrahams et al. 1996).
Tentoxin (Fig. 12.20, 81) was isolated from a
still culture broth of the fungus Alternaria tenuis
as a chlorosis-inducing toxin (Saad et al. 1970). It
is a cyclic tetrapeptide composed of N-methyl-L-
alanine, L-leucine, (aZ)-a,b-didehydro-N-methyl-
phenylalanine and glycine. It inhibits chloroplast
F1-ATPases (CF1s) in sensitive species (e.g. let-
tuce) but not in insensitive species, such as radish
(Linnett and Beechey (1979). It has no effect on
bacterial and mitochondrial F1-ATPases. The KD
value of 81 against lettuce CF1 was 3–5 nM. The
co-crystallization study of spinach CF1 and 81
revealed that 81 bound to the ab interface of the
CF1 in a cleft (Groth 2002). Single molecule studies
of the a3b3g complex of a cyanobacterium, Ther-
mosynechococcus elongatus, with beads attached
on the g subunit suggested that 81 inhibited
ATPase reaction after substrate binding to the bE
subunit by keeping the catalytic site in a closed
conformation with bound 81 (Meiss et al. 2008).
C. Uncouplers
Respiration-dependent ATP synthesis is abolished
by 2,4-dinitrophenol without inhibiting respira-
tion itself. Compounds with this ability are called
“uncouplers”, and they selectively prevent utiliza-
tion of electrochemical proton gradients derived
from respiratory electron transport for net phos-
phorylation of ADP to ATP (Heytler 1979).
To date, some fungal uncouplers have been
reported. ACR-toxin I (ACRL toxin I, Fig. 12.21,
82) is a phytotoxin produced by Alternaria citri
(Gardner et al. 1985) and A. alternata (Kohmoto
et al. 1985). It is a d-lactone, like nafuredin (22),
and 1 mg/ml (2.8 mM) of 82 causes uncoupling of
oxidative phosphorylation and changes in mem-
brane potential in mitochondria from leaves of the
susceptible rough lemon (Citrus jambhiri Lush.,
Akimitsu et al. 1989). Ohtani et al. (2002) discov-
ered the ACR-toxin sensitivity gene (ACRS) in
266 Rokuro Masuma et al.
rough lemon mitochondrial DNA. Though ACRS
was present in the genome of both toxin-sensitive
and–insensitive citrus, the ACRS transcripts of
insensitive plants were shorter than those of sen-
sitive plants. It is suggested that the gene product
of ACRS may be a pore-forming transmembrane
receptor of 82 which leads to uncoupling.
Leucinostatin A (Fig. 12.21, 83) is a nonapeptide
produced by Penicillium lilacinum (Arai et al. 1973)
and some other fungi. It showed an uncoupling
effect against rat liver mitochondria at concentra-
tions above 0.3 mM (Shima et al. 1990). However, 83
also inhibits ATPase activity at a lower concentra-
tion (0.2 mM). It facilitates the transport of monova-
lent and divalent cations with a half-maximal effect
concentration in the range of 0.2–0.8 mM (Csermely
et al. 1994). This ionophoric property of 83 may
cause its uncoupling effect.
VI. Conclusions
Many inhibitors of electron transport and oxida-
tive phosphorylation enzymes have been isolated
from fungal cultures. Some of them, or their ana-
logs, are used as medicines or agrochemicals. The
inhibitors are also highly valuable for elucidating
the mechanism of electron transport and oxida-
tive phosphorylation systems. Specific inhibitors
are used as important tools to study the systems.
Recently, the X-ray crystallography structures of
mitochondrial complexes II, III and IV and the F1
domain of complex V have been reported. As for
complex I, Sazanov and Hinchliffe (2006) reported
CH3
HO
O OH OH
O
ACR-toxin I (82)
CH3 CH3
HO CH3
CH3 O O
H H
H3C N N N N N N
H H
O O
CH3 H
O 3C CH 3
OH
O
Leucinostatin A (83)
CH3
Fig. 12.21. Structures of uncouplers produced by fungi
the crystal structure of the hydrophilic domain
from a bacterium, Thermus thermophilus. Co-
crystallization studies of the complexes and inhi-
bitors clarified the mechanisms of both inhibition
and electron transport.
Over the past few decades, mitochondria
have attracted interest due to their relationship
with various diseases; Leigh syndrome, paragan-
glioma, Parkinson’s disease, Huntington’s dis-
ease, Alzheimer disease and so on (Eng et al.
2003; Wallace 2005). Oxidative phosphorylation
generates reactive oxygen species as toxic bypro-
ducts and they are suggested to cause a wide
range of age-related disorders and various
forms of cancer. The electron transport and oxi-
dative phosphorylation inhibitors are extremely
valuable tools for use in studies of mitochondria-
associated diseases.
Recent research on the respiratory chain of
the helminth Ascaris suum has shown that the
mitochondrial NADH-fumarate reductase system
has an important role in the anaerobic energy
metabolism of adult parasites. Nafuredin (22) is
a potent and selective inhibitor of complex I in
this system, and it showed anthelmintic activity
against Haemonchus contortus in an in vivo study.
A verticipyrone analog (42) was effective against
Echinococcus multilocularis in vitro. Such hel-
minth-specific complex I inhibitors may be good
lead compounds for anthelmintic drugs. A crystal
of adult A. suum complex II (fumarate reductase),
another component of the NADH-fumarate re-
ductase system, was obtained recently (Shimizu
et al. 2007), and analysis of parasite-specific
factors in the enzyme is now in progress. This
CH3 CH3
CH3
OH
CH3
CH3
OH3C CH3 O O CH3 CH3
H H
N N N N
N CH3
H H H
O OH3C CH3
CH3
CH3
 Helminth Electron Transport Inhibitors Produced by Fungi
information and the co-crystallization study of the
enzyme with atpenin A5 (60) may clarify the inter-
action of 60 and helminth complex II and give a
clue to the design of helminth-specific analogs of
60. The first enantioselective total synthesis of atpe-
nin A5 (60) has been achieved recently (Ohtawa
et al. 2009). Thus helminth-specific inhibitors of
elctron transport enzymes are good candidates for
the treatment of heminthic diseases.
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Yonemitsu M, Fukuda N, Kimura T, Komori T, Lindner
HJ, Habermehl G (1989) Crystal structure and NMR
spectrometric analysis of palmarin. Liebigs Ann
Chem 485–487
Zhang Y, Zhu T, Fang Y, Liu H, Gu Q, Zhu W (2007)
Carbonarones A and B, new bioactive g-pyrone and
a-pyridone derivatives from the marine-derived fun-
gus Aspergillus carbonarius. J Antibiot 60:153–157
13 Cyclic Peptides and Depsipeptides from Fungi

HEIDRUN ANKE1, LUIS ANTELO1

CONTENTS dines, promising antifungal drugs against aspergil-

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 losis and candidiasis (Denning 2002; Johnson and
II. Occurrence of Cyclic Peptides and Perfect 2003; Pasqualotto and Denning 2008).
Depsipeptides Within the Kingdom Eumycota Caspofungin derived from pneumocandin and
(True Fungi) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 micafungin derived from FR901379 are examples
1. Siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
2. Diketopiperazines . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 of those novel drugs targeting fungal cell wall syn-
3. Cyclic Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 thesis, e.g. biosynthesis of 1,3-b-glucan (Odds et al.
4. Cyclic Depsipeptides . . . . . . . . . . . . . . . . . . . . . . . . 282 2003; Butler 2004). For a recent review, see
III. Chemical and Biological Diversity of Cyclic Hashimoto (2009). Emodepsin, a semi-synthetic
Peptides and Depsipeptides . . . . . . . . . . . . . . . . . . . . 285 depsipeptide, is used in veterinary medicine against
1. Diversity of Building Blocks . . . . . . . . . . . . . . . . 285
2. Diversity of Structures . . . . . . . . . . . . . . . . . . . . . . 286 helminths (von Samson-Himmelstjerna et al. 2005).
3. Diversity of Biological Activities . . . . . . . . . . . 287 The drug is derived from PF1022A, a metabolite of
IV. Ecological Role of Cyclic Peptides an endophytic fungus from Camellia japonica
and Depsipeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 (Sasaki et al. 1992; Scherkenbeck et al. 2002). As
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 these groups of compounds are well covered in the
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
literature, they are not addressed here in detail.
The biosynthesis of cyclic peptides and depsi-
peptides has attracted the interest of biochemists
I. Introduction since the mid1960s (Gevers et al. 1968). Today, the
focus has shifted from enzymology to genetics,
Cyclic peptides and depsipeptides are widely e.g. the biosynthetic genes and their regulation.
distributed in nature. They are found in plants Therefore Chap. 15 is dedicated to this topic, to
(Gournelis et al. 1998; Tan and Zhou 2006), which the reader is referred.
sponges and other lower sea animals (Bertram A special group of cyclopeptides are the dike-
and Pattenden 2007), cyanobacteria (Welker and topiperazines, which consist of two amino acids
von Döhren 2006), bacteria and fungi alike and linked by two peptide bonds. In the related epipo-
their bioactivities range from antimicrobial, lythiodioxopiperazines the 6-ring is bridged by
insecticidal, nematicidal, antiviral, hepatotoxic, one to four sulfur atoms. The structural diversity
cytotoxic/cytostatic to immunosuppressive and of diketopiperazines (more than 100 different
other pharmacological properties (Kleinkauf and compounds are known from fungi; Buckingham
von Döhren 1997; Pomilio et al. 2006). 2008) is matched by their biological activities.
Some of the peptides and depsipeptides Recently published reviews are available (Cole
produced by fungi have gained entrance into and Schweikert 2003; Gardiner et al. 2005). Inter-
the pharmaceutical market, like cyclosporins estingly, functions in the producing organisms
(Kürnsteiner et al. 2002), ergopeptides (Keller and have been detected for some of these compounds,
Tudzynski 2002), penicillins (Demain and Elander e.g. gliotoxin and related compounds play a role
1999) and cephalosporins (Schmidt 2002), or are as virulence factors in invasive aspergillosis
currently undergoing clinical trials, like the can- (Sugui et al. 2007) and coprogens in host invasion
of plant-pathogenic fungi (Oide et al. 2006;
1
Hof et al. 2007). The reported biological activities
Institute for Biotechnology and Drug Research, IBWF e.V.,
of gliotoxin are very broad and diverse. Anti-
Erwin-Schroedinger-Strasse 56, 67663 Kaiserslautern, Germany;
e-mail: anke@ibwf.de, antelo@ibwf.de
bacterial, antifungal, antiviral, amoebicidal and

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
274 Heidrun Anke and Luis Antelo
immunosuppressive properties have been de-
scribed (see below). Most of these activities are
based on interactions with essential thiol groups
in proteins (Waring and Beaver 1996). Iron che-
lators like dimerumic acid, rhodotorulic acid,
coprogen and its derivatives are involved in iron
uptake (Winkelmann and Drechsler 1997;
Renshaw et al. 2002; Antelo et al. 2006), while
other siderophores, e.g. the hexapeptides ferri-
chrome or ferricrocin, in addition to iron trans-
port or storage functions act as virulence factors
in some human and plant pathogens similar to
coprogens (Howard 1999; Haas et al. 2008).
The group of peptaibiotics, a constantly
growing family of linear a-aminobutyric acid
(Aib)-containing linear peptides has been
enlarged by a small group of cyclic peptides also
containing Aib, now called cyclopeptaibiotics.
Whereas the linear group comprises more than
800 compounds, only nine cyclic compounds
have been reported to date. These are seven tetra-
peptides structurally related to chlamydocin
(Degenkolb et al. 2008) and the scytalidamides,
two heptapeptides containing Aib residues (Tan
et al. 2003).
II. Occurrence of Cyclic Peptides and
Depsipeptides Within the Kingdom
Eumycota (True Fungi)
A. Siderophores
The occurrence and distribution of siderophores
among the taxonomic groups of fungi is very well
covered by the reviews of Renshaw et al. (2002)
and Haas et al. (2008). Zygomycetes very rarely
produce cyclic peptide or depsipeptide sidero-
phores. Up to now the hexapeptide ferrichrysin
seems to be the only example. It is produced by
Cunninghamella blakesleeana (Patil et al. 1995).
The production of diketopiperazine and hexapep-
tide siderophores is common among asco- and
basidiomycetes (Renshaw et al. 2002). The fact
that members of some orders have not yet been
reported to produce siderophores reflects a lack of
investigation rather than presence. There are a
few fungi, however, which do not produce
siderophores: the ascomycetous yeasts Saccharo-
myces cerevisiae and Candida albicans or
Geotrichum candidum and the basidiomycete
Cryptococcus neoformans (teleomorph Filobasi-
diella; Howard 1999; Haas et al. 2008). The inves-
tigation of basidiomycetes is difficult because
iron-free media, which upregulate the biosynthe-
sis of siderophores, often hardly support mycelial
growth, requiring incubation times of eight to ten
weeks (Welzel et al. 2005). In contrast, modern
analytical techniques like HPLC-MSn are sensitive
enough to allow the detection and characteriza-
tion of very small amounts (mg/l of culture). In
addition, as more fungal genomes and NRPS
genes and products become available, it is clear
that siderophores and iron metabolism are impor-
tant virulence determinants (Eichhorn et al. 2006;
Oide et al. 2006; Haas et al. 2008).
It is remarkable that extracellular and intra-
cellular siderophores are not identical and that the
synthesis of intracellular siderophores is often not
iron-dependent.
As an example, most Trichoderma species excrete copro-
gen-type siderophores and ferricrocin for the capture
and transport of iron and use palmitoylcoprogen located
within the mycelia as storage compound. In T. pseudo-
koningii and T. longibrachiatum however, palmitoylco-
progen was not detected, but these two species excrete
fusigen-type siderophores in addition to coprogen and
fericrocin (Anke et al. 1991). Magnaporthe grisea uses
intracellular ferricrocin for iron storage and under iron
deprivation excretes four coprogen derivatives (Hof et al.
2007). In other plant-pathogenic fungi like Fusarium
graminearum, F. culmorum, F. pseudograminearum,
Cochliobolus heterostrophus and Gibberella zeae ferricro-
cin has also been reported as intracellular siderophore
(Oide et al. 2007; Tobiasen et al. 2007). The situation in
the human pathogen Aspergillus fumigatus is similar.
Ferricrocin is located in the mycelia, a hydroxylated
derivative in the conidia and triacetylfusigen is excreted
(Schrettl et al. 2007).
The structures of several iron-free siderophores,
e.g. rhodotorulic acid, 2-N-methylcoprogen, pal-
mitoylcoprogen, ferricrocin and ferrichrome are
given in Fig. 13.1.
B. Diketopiperazines
Simple diketopiperazines may be detected in fer-
mentations of many fungi. Sometimes it is diffi-
cult to decide whether these are degradation
products of proteins and peptides or synthesized
de novo (Prasad 1995). In the future, this problem
might be solved by molecular genetics, since the
presence of the relevant biosynthetic genes can be
 Cyclic Peptides and Depsipeptides from Fungi 275

O Fig. 13.1. Structures of some

H
N intracellular and extracellular
N OH
HO siderophores produced by fungi
O OH
O O

O O

N NH O
OH HN N
OH
Desferripalmitoylcoprogen O

O O
O
O N
HO NH NH
OH
N
OO O O O
O O
HN
O N
NH
NH OH O
N OH HO N
O
O
O
Desferritriacetylfusigen O
HO
O OH
H H N
O
O N
OH N
O OH
N OH N
N O
H H
O O
Desferri-2-N-methylcoprogen
Rhodotorulic acid

O O O O
H H
N HO N
N N N N
H H
O O HN O OH
O HN OH O NH OH
O NH O OH O
H H
N N N N
N O N O
H H
O O O O
N OH N OH

Desferriferrichrome Desferriferricrocin

proof of de novo synthesis (Chap. 15). The recently and neoechinulin from an marine Aspergillus
demonstrated behavioral effects and occurrence in species (Li et al. 2004). As in many other cases,
humans of cyclo(His-Pro) stimulated research on the simple alkaloid is the biogenetic precursor of
such compounds which are easily accessible by the other two compounds. With antimicrobial,
chemical synthesis. However, cyclo(His-Pro) has cytotoxic, phytotoxic, insecticidal and other test
not yet been reported from fungi. This may be due systems which have been extensively used in
to the fact that its bioactivities, e.g. inhibition of screenings for bioactive natural products, simple
food intake and inhibition of prolactin secretion diketopierazines are less frequently detected. One
or modulation of pain perception (Prasad 1995) example is the fungistatic mactanamide from a
are not suited for a screening of microbial cul- marine Aspergillus species (Lorenz et al. 1998).
tures. Usually these compounds are detected dur- Simple diketopiperazines have been described
ing the isolation of other metabolites and from hetero- and homobasidiomycetes, for
described as side-products. A recent example is example Ustilago cynodontis, Entoloma haastii
L-alanyl-L-tryptophan anhydride isolated together and Stereum hirsutum (Turner and Aldridge
with golmaenone, a radical scavenger compound, 1983), ascomycetes like Rosellinia necatrix,
276 Heidrun Anke and Luis Antelo

Claviceps species, Eurotium and Emericella spe-
cies, Leptosphaeria species including their ana-
morphs, Aspergillus, Phoma and Coniothyrium
species (Turner and Aldridge 1983; Cole and
Schweikert 2003; Blunt et al. 2006).
Aspergillus and Penicillium species are very prolific pro-
ducers of cyclic dipeptide-derived mycotoxins like fumi-
tremorgins, verruculogens or roquefortine C, while
sporidesmins, mycotoxins that cause facial eczema in
grazing sheep, are produced by Pithomyces chartarum
(Betina 1989). From several Penicillium species, mycelia-
namide, one of the very “old” diketopiperazines, has been
known since 1931. This compound was detected during
early screenings after the discovery of penicillin G. The
recently described sulfur-containing gliovictin was
obtained from an endophytic Penicillium janczewskii
(Gunatilaka 2006) and diketopiperazine-derived rostra-
tins from a marine Exserohilium rostratum (Tan et al.
2004). To the long list of Penicillium species producing
diketopiperazines, P. dipodomyis, P. nalgiovense, P. fell-
utanum and P. simplicissimum were recently added
(Lewis 2002).
Examples for structures of simple and complex
diketopiperazines are found in Fig. 13.2.
From cultures of a number of fungi produc-
ing cyclic depsipeptides, e.g. Beauveria bassiana,
dipeptides composed of the amino acids
occurring in the depsipeptides have been

H
O N
OH
H
O N
N O
H H
O N
N O N
HO H
O NH N O
Mactanamide L-Alanyl-L-tryptophan anhydride H
O

O O
Golmaenone
NH NH

HN HN
N N
O H
H
O O
Neoechinulin A
Neoechinulin
O
OH
N
N H O O
O HO OH
Mycelianamide O N
HO O N
OH O N
N H
O
N O O
O N
H
O
O O
Fumitremorgin A
OH
Verruculogen O
H N
O N H
N
O O
O
NH
N N N
N
H H
O HN

HN Ergovaline
Roquefortin C

Fig. 13.2. Structures of some diketopiperazines

 Cyclic Peptides and Depsipeptides from Fungi 277

isolated. Other insect pathogens like Verticillium Epipolythiopiperazines with more than 60
species and Metarhizium anisopliae as well as members, gliotoxin being the most prominent,
plant-pathogenic fungi, e.g. Colletotrichum are widely distributed in nature. Their pro-
gloeosporioides, Exserohilum holmi, Gliocladium ducers are mainly found among the ascomycete
deliquenscens, Alternaria and Trichoderma pro- genera Aspergillus, Penicillium, Gliocladium, Ver-
duce dipeptides. An unidentified endophyte from ticillium, Chaetominum, Emericella, Acrostalag-
mangrove leaf produces two cyclic depsipeptides mus (syn. Verticillium), Pithomyces, Bionectria,
and three diketopiperazines (Huang et al. 2007). Leptosphaeria, Hyalodendron, Trichoderma, Siro-
The role of the compounds, dipeptides and desmium (syn. Coniosporium), Epicoccum, Ara-
depsipeptides, in insect and plant-pathogenicity chniotus and Pseudallescheria (Turner and
has not yet been completely elucidated. As mo- Aldridge 1983; Betina 1989; Takahashi et al.
lecular tools become more easily available, 1994; Gardiner at al. 2005; Li et al. 2006; Zheng
this question might be addressed or even an- et al. 2007). There is one report on the occurrence
swered in the near future, especially since the of an epipolythiopiperazine in lichens, e.g.
elucidation of the ecological function of second- Xanthoparmelia scabrosa (Ernst-Russell et al.
ary metabolites for the producers becomes more 1999). As is true for many lichen metabolites,
interesting (see below). it may be also in this case the ascomycetous fungal

OH
Cl OH
O
O
O O N N
OH H S
SN O
O NS
N S N OH O H
S OH SN
OH O
O Sporidesmin A
Gliotoxin OH
Epicorazine C O
OH
O
O O
H N
OH S
H H
S
N N N S
S O
N
HO
H
O
O
O O
Scabrosin S N N
H H S
N SN
O
N OH Chaetocin
S OH
O
Gliovictin
OH O OH
H
O H
N N
S N N

O N
S O
S OH O
N O
O
S
O N N (S)4 N
N
H H
OH
O
Vertihemiptellide A Leptosin I

Fig. 13.3. Structures of some epipolythiopiperazines

278 Heidrun Anke and Luis Antelo

partner which is responsible for the production C. Cyclic Peptides

of scabrosin. The production of epicorazine C by
Stereum hirsutum, a basidiomycete, seems a bit
questionable since related epicorazines are pro-
duced by Epicoccum nigrum and E. purpurascens
(Kleinwachter et al. 2001). Overlaps between
metabolites from basidiomycetes and ascomy-
cetes are fairly rare but do occur occasionally.
Other examples may be beauvericin and
chlamydocin (see below). The structures of glio-
toxin, epicorazines, scabrosin, vertihemiptellide A
and other epipolythiopiperazines are given in
Fig. 13.3.
Cyclic peptides are mainly produced by ascomy-
cetes and their anamorphs. Among cyclic pep-
tides, the immunomodulating cyclosporins
constitute the largest group with 46 members.
The producing organisms are found mainly in
the ascomycetous families Hypocreaceae and
Clavivipitaceae and their anamorphs Tolypocla-
dium inflatum, T. tundrense and T. terricola. In
addition, three soil-borne insect pathogens, Neo-
cosmospora vasinfecta, Acremonium luzulae, a
Cyclindrotrichum species, Stachybotrys chartarum,

O
O N
N HN
H NH
N NH O
N O OO
H
N O O
O HN O
O NH
O N
N
O
Apicidin

Scytalidamide

O
H H
O N
N N
H H
N
O
O HN O
O H
O HN HN
N H
N
H O
O
HN

CJ-15,208

OH
O
H H
Cycloaspeptide A N
N
O H
H H
NH
H H
N
O N O
H H
O
S S

Malformin C

Fig. 13.4A. Structures of some simple cyclopeptides

 Cyclic Peptides and Depsipeptides from Fungi 279

OH
H H
OH
O O
H
O HN N
N H H
H NH N N
O
O
NH O
– +
HN
O S N OH O
O H N
H O HO
H2N H O H
N N NH O N HN O
HO N O
H NH
O O N O N

α-Amanitin HO

Argadin
O
H
N N
O N N
O O
O N
NH
O
O
N O N
N O O
H
H OH
N N O
N N O O
O N N
H H
O
Omphalotin A
O N
H
N
O HO N
H H
N N N O
O N Cl Cl

O O
O N Cyclochlorotine
O
N O
HO
O
OH
NH N O N
O O O
H
N N
N N O
O

O O
Omphalotin C
OH

Fig. 13.4B. Structures of some complex cyclopeptides.

Trichoderma viride, a Leptostroma anamorph of mycotoxins even so they are rarely found in food or feed
stuff.
Hypoderma eucalyptii, Chaunopycnis alba and an
unidentified mycelium sterilium have been The antifungal echinocandins comprising differ-
reported to produce cyclosporins (Matha et al. ent compounds (aculeacin A, echinocandin B,
1992; Traber and Dreyfuss 1996). The structure of pneumocandins, mulundocandins, FR901379,
cyclosporin A is found in Fig. 13.4C. Figure. 13.4A WF11899A, B, C, FR227673, FR190293, etc.) have
been reported from several Aspergilli, Coleo-
shows examples of simpler cyclospeptides.
phoma empetri, C. crateriformis, Chalara species,
The malformins, a group of nine phytotoxic compounds,
Tolypocladium parasiticum and Zalerion arbori-
are only found within the Aspergillus niger group (Kobbe cola (Iwamoto et al. 1994b; Anke and Erkel 2002;
et al. 1977). Some authors classify the compounds as Denning 2002; Kanasaki et al. 2006a, b, c,).
280 Heidrun Anke and Luis Antelo

O
NH O
O HN NH
N N N
O H H H
O

HO HN N
HO
OH
O H
N N
H O N
O O
OH NH
O NH2
HN O
Argifin
OH HN OH
O HN O O

Serinocyclin A
N O
OH H

O O
N
HO O
O O
H
N N N
N N
N
H O
O O
O N
O
N NH
O
N H O
H N
N
N N
O H O O
O

Cyclosporin A Psychrophilin A

Fig. 13.4C. Structures of other complex cyclopeptides

The Zalerion strain producing echinocandin B was later
reclassified as Glarea lozoyensis, a new anamorph genus
and species within the Leotiales (Bills et al. 1999). The
fungus producing arborcandins (Ohyama et al. 2000), has
not been identified.
The structures of some of these compounds can
be found in Fig. 13.5.
Producers of various cyclic peptides are found
in many other families and genera, for example
Diheterospora, Gliocladium, Cylindrocarpon,
Clonostachys, Cochliobolus and Fusarium (Lewis
2002; Adachi et al. 2005; Weber et al. 2006;
Degenkolb et al. 2008).
As endophytic fungi have recently come
into focus as producers of bioactive natural com-
pounds, it is not astonishing that also novel cyclic
peptides have been reported from these fungi.
A pentapeptide was isolated from an unidentified endophyte
from the seed of Avicinnia marina (Gunatilaka 2006), other
cyclopeptides from endophytic Fusarium species (Shiono
et al. 2007), Epichloe typhina (Seto et al. 2007) or endophyte
“2221” from Castaniopsis fissa (Yin et al. 2005). More than
450 cyclic peptides are known from plants (Tan and Zhou
2006); some of these actually may be produced by endophyt-
ic fungi in planta.
In recent years, marine habitats have drawn much
interest as ecological niches for producers of novel bioac-
tive metabolites. The unguisins were isolated from a ma-
rine-derived strain of Emericella unguis (Malstrom 2002).
Among cyclic peptides from obligate marine ascomycetes
are the highly cytotoxic trapoxin A produced by Corollos-
pora intermedia (Daferner 2000) or scytalidamides from a
Scytalidium species from a marine alga (Tan et al. 2003).
JM47, structurally related to HC-toxins and trapoxin, was
isolated together with enniatin from a marine-derived
Fusarium species (Jiang et al. 2002). Trapoxins are also
known from terrestrial fungi, e.g. Helicoma ambiens, the
anamorph of Thaxteriella pezicula (Itazaki et al. 1990)
and structurally related metabolites have been described
from the phytopathogenic Cyclindrocladium scorparium
(teleomorph Calonectria morganii) and Cochliobolus car-
bonum (Degenkolb et al. 2008).
For structures see Fig. 13.6.
The only cyclopeptides, besides the sidero-
phores, known from submerged cultures of basi-
 Cyclic Peptides and Depsipeptides from Fungi 281

HO OH
HO HO O
O
N
H
N
N H H
NH2 OH O
HN
O O
O H
H NH N
H H
O H N OH HO
N
H
H H O N
HO OH HN O
OH O H
OO
Pneumocandin D0 O O
NH
O

OH HO HO OH LL15G256γ OH
O
NH
OH N
N H O
OH O
HN O
O O O S OH
NH O
H
HO O H HN OH
N
OH HO HO
H O
O
OH O NH2
N N
OH H
HO O HN
O
Echinocandin B O OH
NH N
O
O
H
N
N
H OH
WF11899 C OH O

HO
OH
O O
H H
N N N
N N
H H
O O O O O
OH HN O
H 2N O OH
NH O O
H H
N N NH
O N N
H FR901469
O O
OH OH HO OH

H2N O

Fig. 13.5. Structures of some b-1,3-glucan synthase inhibitors

diomycetes are the omphalotins from Omphalotus
olearius (Büchel et al. 1998a,b), amanitins from
Amanita exitialis (Zhang et al. 2005) and chlamy-
docins from a Peniophora strain isolated from
soil (Tani et al. 2001). The chlamydocins are
tetrapeptides with Aib and an unusual amino
acid. Most of these are produced by ascomycetes,
e.g. Diheterospora chlamyydosporia (Closse and
Huguenin 1974) and V. coccosporum (Gupta
et al. 1994). Interestingly, the omphalotins
produced by a monokaryotic strain differ from
those found in the dikaryotic parental strain (Lier-
mann et al. 2009). However, all O. olearius strains
irrespective of their geographical origin produce
omphalotin derivatives (Anke et al., unpublished
data). In fruiting bodies omphalotins could not
282 Heidrun Anke and Luis Antelo

Fig. 13.6. Structures of some

histone deacetylase inhibitors

H
O N
O

O HN O N
NH N H
N
O
NH HN O O
O
OH
O
O
Chlamydocin FR235222 O

O
O O

O O
O NH N N
H
N
NH
NH HN O O H
O N
O O
O

Trapoxin A HC-toxin I

H O
N

O NH N
H
N
O
O
OH
O
JM47

be detected, contrary to Amanita exitialis anamorphs Helminthosporium and Bipolaris,

carpophores which contained tenfold more a-
and b-amanitin as compared to the slow growing
mycelial cultures (Zhang et al. 2005). For recent
surveys of Amanita toxins from fruiting bodies
see Li and Oberlies (2005), Liu (2005) and Pomilio
et al. (2006). Figure 13.4B shows the structures of
omphalotins and a-amanitin.
D. Cyclic Depsipeptides
Most depsipeptides are metabolites from ascomy-
cetes and their anamorphs. They are widespread
in phytopathogens (e.g. Cochliobolus with
Calonectria and its anamorph Cyclindrocladium,
as well as Fusarium and Alternaria), insect patho-
gens (Aschersonia, Beauveria, Cordyceps, Diheter-
ospora, Fusarium, Hirsutella, Isaria, Metharizium,
Paecilomyces, Verticillium) and others (Zimmer-
mann 2007a, b; Buckingham 2008). For a compi-
lation of beauvericins and enniatins produced by
Cordyceps species and their anamorphs as well as
other insect pathogens see Isaka et al. (2005a, b).
Figure 13.7 gives the structures of some cyclodep-
sipeptides.
Up to now the pteratides (Fig. 13.7C) are
the only depsipeptides reported from basidio-
mycetes, namely from the fruiting bodies of a
 Cyclic Peptides and Depsipeptides from Fungi 283

O O
O H NH
N O
N N
NH
H O O
O O
NH
N O O O
H O N
H O
Destruxin A AM toxin I
O

HN
O HN O O

NH
O OH O O
HN O O
O O O
O NH
Icosalide A1
HN
N
H Beauverolide II
HO
O O
H
O N O O
N
H N O
NH O
NH
O N
O
O N O
H O
O O N
O
O

Isariin A Beauvericin

O O
O N
O
O O O
O
O N N
N
O O
O O
N
N
O O
N O O
O O
O

Bassianolide Enniatin I

Fig. 13.7A. Structures of some simple cyclodepsipeptide

Pterula species (Chen et al. 2006). From zygomy-
cetes none have been described. One report on
the production of beauvericin by Laetiporus sul-
fureus (Badan et al. 1978) could not be confirmed
by other groups. In our cultures from L. sulfureus
from different locations we could only detect
laetiporic acid and its derivatives (Davoli et al.
2005).
Since the review of Anke and Sterner (2002),
additional producers of bioactive depsipeptides
have been reported, for example marine-derived
strains of Beauveria fellina (Lira et al. 2006),
Verticillium sp. FKI-1033 (Monma et al. 2006),
Aspergillus carneus (Capon et al. 2003),
Torrubiella luteorostrata and its anamorph Paeci-
lomyces cinnamomeus (both isolated from a scale
insect; Isaka et al. 2007), Verticillium hemipteri-
genum (Supothina et al. 2004), an Aureobasidium
species from the tropical rain forest (Boros et al.
2006), an unidentified endophytic fungus (Huang
et al. 2007) and a soil-borne Phoma species
(Aoyagi et al. 2007). Pseudodestruxins have been
284 Heidrun Anke and Luis Antelo

O
O O
N
O HN NH
O H
N
O O H
O H O
O HN
H H
O N
O
O N H
O O
O N
O
O
Sansalvamide A
PF1022A
O
O
N N
O NH O
O NH

OO
N
HN N
O
N
O O
H H
O N O
Pestahivin O N O
HN
N N
O N

O O N
OH O
O O O
N N NH OH
N N
O O
O O
H Petriellin A
N N N N O
O O

O
OH
O O
H NH
N N
N
O Aureobasidin A
O

Fig. 13.7B. Structures of some complex cyclodepsipeptides

reported from Nigrosabulum globosum (Che et al. One of the few aquatic fungi investigated
2001) and reviews on destruxins and the produc- for secondary metabolite production is Clavar-
ing organisms have been published by (Pedras iopsis aquatica from which the antifungal cla-
et al. 2002) and Zimmermann (2007b). variopsins A and B were isolated (Kaida et al.
The endophyte-producing PF1022A (and 2001).
related anthelmintic cyclooctadepsipeptides) iso- Analogues of the lipopeptides with 1,3-b-glu-
lated from leaves of a camellia has been identified can synthase inhibitory activity are the lipodepsi-
based on its 18S rRNA gene sequence as a mem- peptides FR901469 or LL15G256g (see Fig. 13.5).
ber of the Xylariaceae close to Xylaria polymorpha The former is produced by an unidentified fungus,
and Rosellinia necatrix (Miyado et al. 2000). the latter (identical to arthrichitin from Arthrinum
 Cyclic Peptides and Depsipeptides from Fungi 285

OH
H H
O N N O
HO
O O
HN
O O O OH
HN O
NH O NH2
N
O OH HN
O O
O N
O O O H
NH2
O N O OH
O
N Glomosporin
O
N
O O O
O

Verticilide O

O OHO HN
O O
O O
N NH
O O H
O
N O OH
HN N N
H O Stevastelin B
O O
O
N N
O
N
O N N O
H
O
OH
Clavariopsin A O N
O

O N
NH
O
O N N O
O O

O N
N
Pteratide I
HN O
O

Fig. 13.7C. Structures of other complex cyclodepsipeptides

spaeospermum) by Hypoxylon oceanicum (Abba- structural diversity. This diversity is brought

nat et al. 1998; Fujie et al. 2000). upon by the different building blocks in the ring:
proteinogenic amino acids including their D-iso-
mers, nonproteinogenic amino acids, branched or
III. Chemical and Biological Diversity unbranched lipoamino acids and hydroxylated
of Cyclic Peptides and Depsipeptides short-, medium- and long-chain fatty acids. The
diversity of the building blocks can be deduced
A. Diversity of Building Blocks from Tables 13.1–13.3, which give a compilation
of unusual building blocks (Table 13.1 unusual
Cyclic peptides and depsipeptides constitute a amino acids, Table 13.2 unusual fatty acids) and
class of natural compounds with an enormous various modifications (Table 13.3).
286 Heidrun Anke and Luis Antelo

Table 13.1. Diversity of amino acid building blocks in cyclic peptides and depsipeptides

Amino acid Example Figure Reference

a-Aminoadipic acid Argadin 13.4B Arai et al. (2000)
Prolyl-homoserine Argadin 13.4B Arai et al. (2000)
b-Keto tryptophan LL15G256g 13.5 Abbanat et al. (1996)
Propylleucine Pestahivin 13.7B Hommel et al. (1996)
Dehydroalanine AM-toxin I 13.7A Ueno et al. (1975)
a-Amino-p-methoxyphenylvaleric acid AM-toxin I 13.7A Ueno et al. (1975)
N5-Hydroxyornithine Siderophores 13.1 Renshaw et al. (2002)
4-Methylproline FR-235222 13.6 Mori et al. (2003)
2-Butenyl-4-methylthreonine Cyclosporin A 13.4C Rüegger et al. (1975)
a-Aminobutyric acid Cyclosporin A 13.4C Rüegger et al. (1975)
3-Hydroxyhomotyrosine WF-11899C 13.5 Iwamoto et al. (1994b)
5-Hydroxyornithine WF-11899C 13.5 Iwamoto et al. (1994b)
Dichloro-proline Cyclochlorotine 13.4B Yoshioka et al. (1973)
b-Phenyl-b-aminopropionic acid Cyclochlorotine 13.4B Yoshioka et al. (1973)
b-Alanine Destruxin A 13.7A Rees et al. (1996)
b-Aspartic acid Argifin 13.4C Arai et al. (2000)
a-Aminoisobutyric acid Chlamydocin 13.6 Closse and Huguenin (1974)
Isovaline FR-235222 13.6 Mori et al. (2003)
1-Aminocyclopropane-1-carboxylic acid Serinocyclin A 13.4C Krasnoff et al. (2007)
Pipecolinic acid Trapoxin A 13.6 Itazaki et al. (1990)
Anthranilic acid Psychrophilin D 13.4C Dalsgaard et al. (2005)
2-Amino-8-oxo-9-hydroxydecanoic acid JM47 13.6 Jiang et al. (2002)
2-Amino-9,10-epoxy-8-oxodecanoic acid HC-toxin I 13.6 Gross et al. (1982)

Table 13.2. Diversity of hydroxyacid building blocks in cyclic depsipeptides

Acid Example Figure Reference

2-Hydroxyisovaleric acid Clavariopsin A 13.7C Kaida et al. (2001)
3,4-Dihydroxy-4-methylhexadecanoic acid Glomosporin 13.7C Ishiyama et al. (2000)
2-Hydroxy-3-methylpentanoic acid Enniatin I 13.7A Nilanonta et al. (2003)
2-Hydroxyheptanoic acid Verticilide 13.7C Monma et al. (2006)
2-Hydroxy-4-metylpentanoic acid Sansalvamide A 13.7B Belofsky et al. (1999)
Phenyllactic acid PF1022A 13.7B Sasaki et al. (1992)
Lactic acid PF1022A 13.7B Sasaki et al. (1992)
3-Hydroxydodecanoic acid Isariin A 13.7A Wolstenholme and Vining (1966)
3-Hydroxy-4-methyldecanoic acid Beauverolide II 13.7A Mochizuki et al. (1993)
3-Hydroxydecanoic acid Icosalide A1 13.7A Boros et al. (2006)
3,5-Dihydroxy-2,4-dimethylstearic acid Stevastelin B 13.7C Morino et al. (1994)
2-Hydroxy-4-cyanobutyric acid Pestahivin 13.7B Hommel et al. (1996)
2-Hydroxy-4-enoylpentanoic acid Destruxin A 13.7A Rees et al. (1996)
2,4-Dimethyl-3-hydroxydodecanoic acid LL15G256g 13.5 Abbanat et al. (1996)

B. Diversity of Structures Cyclic peptides including the cyclosporins are

Additional variations are due to the different
numbers of building blocks, their arrangement
(e.g. sequence in the ring) and their linkage (e.g.
amide and ester bonds). Some depsipetides like
the enniatins, beauvericins, bassianolide or verti-
cilide show a symmetric arrangement in the ring.
The majority however are asymmetric, like the
destruxins, beauverolides, isariins or Alternaria
toxins (Fig. 13.7A,C).
asymmetric, as are the echinocandins. The num-
ber of building blocks in cyclic peptides varies
from two in the diketopiperazines, some of
which are symmetric if composed of two residues
of the same amino acid, to 12 in the omphalotins,
which are at present the largest cyclopeptides
known from fungi. In addition, the omphalotins
are an example of modifications after ring closure.
Omphalotins B, C and D are derived from ompha-
lotin A by hydroxylation followed by acylation to
 Cyclic Peptides and Depsipeptides from Fungi 287

Table 13.3. Modifications in cyclic peptide and depsipeptides

Modification/substitution Example Figure Reference

O-Methyl Clavariopsin A 13.7C Kaida et al. (2001)
N-Methyl Omphalotin A 13.4B Sterner et al. (1997)
Methoxy Pestahivin 13.7B Hommel et al. (1996)
Acetyl Omphalotin C 13.4B Büchel et al. (1998a)
3-Hydroxy-methylbutanoyl Omphalotin C 13.4B Büchel et al. (1998a)
Palmitic acid WF-11899C 13.5 Iwamoto et al. (1994b)
3-Hydroxypalmitic acid FR 901469 13.5 Fujie et al. (2000)
Linoleic acid Echinocandin B 13.5 Keller-Juslen et al. (1976)
Sulfate WF-11899C 13.5 Iwamoto et al. (1994b)
Nitro Psychrophilin A 13.4C Dalsgaard et al. (2005)
Halogenation Sporidesmin A 13.3 Fridrichsons and Mathieson (1962)
Isoprenyl Roquefortine C 13.2 Scott et al. (1979)
Prenyl Fumitremorgin A 13.2 Eickman et al. (1975)
Geranyl Mycelianamide 13.2 Birch et al. (1956)
N-Methylcarbamoyl Argifin 13.4C Arai et al. (2000)
Hydroxylation
3-Hydroxyvaline Omphalotin C 13.4B Büchel et al. (1998a)
4,5-Dihydroxyornithine Echinocandin B 13.5 Keller-Juslen et al. (1976)
3,4-Dihydroxyhomotyrosine Echinocandin B 13.5 Keller-Juslen et al. (1976)
2,6-Dihydroxyphenylalanine Mactanamide 13.2 Lorenz et al. (1998)
3,4-Dihydroxyproline Pneumocandin D0 13.5 Morris et al. (1994)

the corresponding esters and formation of addi- N-hydroxylations followed by an acylation

tional ring structures (Büchel et al. 1998).
Novel omphalotins were recently isolated from a mono-
karyotic strain. The elucidation of their structures was
greatly hampered by their instability (Liermann et al.
2009). These omphalotins bear additional hydroxyl
groups, thus bringing the number of known cyclic pep-
tides from O. olearius to 11. A second hydroxylation at the
tryptophan leads to a novel ring system (Fig. 13.4B).
HPLC-MS spectra of enriched extracts indicate the pres-
ence of additional members of the group. The psychrophi-
lins are nitropeptides with unusual structures (Fig. 13.4A).
The compounds are produced by several psychrotolerant
Penicillium species (Dalsgaard et al. 2004a, b; 2005).
Cyclochlorotine, a mycotoxin from P. islandicum contains
a dichloroprolyl residue (Betina 1989).
The depsipeptides start with four building blocks
(angolide, beauverolides) up to 12 in the antibiotic
FR901469 (a member of the 1,3-b-glucan synthase
inhibitors; Fujie et al. 2001) and 13 in petriellin A
(Lee et al. 1995). The latter contains b-phenyllactic
acid, a building block not often found in cyclopep-
tides and -depsipeptides. Further modifications
of cyclic peptides and depsipeptides include
N-methylation, hydroxylations, acylation, isopre-
nylation and the introduction of sulfate-, nitro-
chloro- or cyano- groups. These modifications
can occur at the beginning of biosynthesis, like
N-methylations, or after cyclization, e.g. C- and
(Glinski et al. 2001; Chap. 15). In many cases how-
ever it is not clear at which step the modifications
occur. The low substrate specificity of the NRPS
enzymes allows the incorporation of modified ring
components. In fact, Zocher and his group have
made use of this to produce novel enniatin deriva-
tives in vitro (Feifel et al. 2007).
C. Diversity of Biological Activities
The structural diversity of diketopiperazines,
cyclopeptides and -depsipeptides is matched by
the diversity of their biological activities. To list
all activities and compounds would be beyond of
the scope of this chapter. An overview on
biological activities of diketopiperazines is given
by Martins and Carvalho (2007), cyclic depsipep-
tides and their biological activities are reviewed
by (Sarabia et al. 2004), while insecticidal and
other biological activities of destruxins, isariins,
enniatins, and beauverolides are reviewed by
Anke and Sterner (2002) and by Zimmermann
(2007a, b). Some of the compounds exhibit rath-
er selective activities like the antifungal, 1,3-b-
glucan synthesis inhibitors (see below) whereas
others like gliotoxin show a broad spectrum of
activities. While the former (due to fewer side-
288 Heidrun Anke and Luis Antelo
effects) generally have a higher potential to be
developed into drugs or pesticides, the latter
might be of interest as biochemical tools or
chemical building blocks. In the following, we
attempt to give an overview on the different
biological activities exhibited by fungal cyclopep-
tides and -depsipeptides.
Gliotoxin, already isolated in 1932, recently
regained interest not only due to its immunosup-
pressive and apoptosis-inducing activities (Waring
et al. 1988) but moreover due to its occurrence in
the blood of aspergillosis patients and its effects on
various human cells among them an inhibition of
cell adherence in macrophages (Amitani et al. 1995;
Kamei and Watanabe 2005). The plethora of
biological activities is evident from the number of
papers published on gliotoxin and related epipo-
lythiodioxopiperazines (Waring and Beaver 1996;
Hume et al. 2002; Gardiner et al. 2005).
The vertihemiptellides A and B and their S-methylated
monomers exhibit antimycobacterial and cytotoxic effects
(Isaka et al. 2005b). Sirodesmin PL produced by Lepto-
sphaeria maculans has phytotoxic, antibacterial and insec-
ticidal properties (Rouxel et al. 1988; Boudart 1989) and
the leptosins inhibited the proliferation of P388 lympho-
cytic leukemia cells with an ED50 of 1.1–1.3 mg/ml (Taka-
hashi et al. 1994).
The HC-toxins, host-specific toxins from Coch-
liobolus carbonum (anamorph Helminthosporium
carbonum), are cyto- and phytotoxic and inhibitors
of histone deacetylase (Taunton et al. 1996).
Structurally related tetrapeptides (Fig. 13.6) like apicidin
from a Fusarium species (Darkin-Rattray et al. 1996; Singh
et al. 2002), JM47 from a marine Fusarium species (Jiang
et al. 2002), FR235222 from an Acremonium species (Mori
et al. 2003) or the chlamydocins from Diheterospora chla-
mydosporia (Closse and Huguenin 1974) and Peniophora
sp. (Tani et al. 2001) have been reported to exhibit anti-
protozoal activity, to induce apoptosis, to have immuno-
suppressive effects or to retard plant growth (de Schepper
et al. 2003).
Due to their toxic effects in animal and humans and
their occurrence in food and feedstuff, fumitremorgins,
verruculogens, roquefortins C and D, sporidesmins, chae-
tocin, cyclochlorotine and malformins were classified as
mycotoxins (Betina 1989). For their different biological
activities the reader is referred to the vast online literature
on this group of fungal products.
Malformin C (Fig. 13.4), despite its antibacterial,
plant-deforming and fibrinolytic activities, recently
aroused some interest due to its inhibitory effects
on bleomycin-induced G2 arrest, thus potentiat-
ing its DNA-damaging action, a mode of action
that might be useful for the treatment of cancer
(Hagimori et al. 2007).
Cyclosporins are not the only immunomodu-
lating fungal metabolites. Many epipolythiodiox-
opiperazines, in addition to other biological
activities, are immunosuppressants.
Sevastelins, cyclodepsipeptides with a lipophilic
side-chain, from a Penicillium species blocked human T
cell activation in vitro and showed low acute toxicity in
mice (Morino et al. 1994). HUN-7293 acts as inhibitor of
cytokine-induced expression of vascular cell adhesion
molecule-1 on human endothelial cells (Hommel et al.
1996). It is structurally identical to pestahivin.
The depsipeptide aureobasidin A has an interest-
ing mode of action, the inositol phosphoceramide
synthase (IPS). The fungal enzyme is considered to
be an attractive target for novel fungicides. Further
development of aureobasidin A was hampered by
its inhibitory effects on ABC transporters in yeasts
and humans (Fostel and Lartey 2000). The pleo-
fungins from a Phoma species showed antifungal
activity towards Candida albicans, Cryptococcus
neoformans and A. fumigatus with minimal inhib-
itory concentrations in the range of 1 mg/ml or
lower (Yano et al. 2007). The compounds inhibited
the A. fumigatus IPS with IC50 values of 1 ng/ml
(Aoyagi et al. 2007).
Neoechinulin A has protective activity in
PC12 cells against lethal effects of peroxynitrite
and against 1-methyl-4-phenylpyridine, a neuro-
toxin capable of inducing neurodegeneration in
humans (Kajimura et al. 2008). The cyclic tetra-
peptide CJ-15,208 is a kappa opinoid receptor
antagonist (Saito et al. 2002) and four depsipep-
tides were reported to be selective and competitive
human tachykinin receptor antagonsits (Hedge
et al. 2001).
Among nine beauverolides tested for acyl-CoA:
cholesterol acyltransferase (ACAT) inhibitory ac-
tivity in CHO-cells expressing ACAT1 or ACAT2,
beauverolides I and III inhibited ACAT1 rather
selectively, no antimicrobial or cytotoxic activities
were detected and beauvericin was cytotoxic (Mat-
suda et al. 2004; Ohshiro et al. 2007). ACAT is
discussed as a target for new antiatherosclerotic
agents (Roth 1998; Namatame et al. 2004).
The outstanding anthelmintic activity of
PF1022A combined with its mode of action, e.g.
binding to the latrophilin-like receptor of
 Cyclic Peptides and Depsipeptides from Fungi
Haemonchus contortus (Conder et al. 1995; Saeger
et al. 2001) and low toxicity led to the develop-
ment of emodepsin, a novel drug used in animal
health.
Antiparasitic properties have been reported
for cycloaspeptides A and D (Dalsgaard et al.
2004b). Verticilide, a cyclic depsipeptide isolated
from the culture broth of Verticillium sp. FKI-
1033, inhibits the binding of ryanodine to the
receptor (RyR) and has insecticidal activity
(Monma et al. 2006). Serinocyclin A isolated
from M. anisopliae condia produced a sublethal
locomotory defect in mosquito larvae (Krasnoff
et al. 2007). Argifin and argadin, two cyclopenta-
peptides from a Gliocladium and a Clonostachys
species, are potent inhibitors of chitinase B from
Serratia marcescens (Houston et al. 2002). When
injected into cockroach larvae, the moult was
arrested. Besides cyclopeptides and -depsipep-
tides fungi also produce other peptides with
insecticidal activities, recent examples are the
neofrapeptins from Geotrichum candidum
(Fredenhagen et al. 2006). Selective nematicidal
properties have been reported only for the
omphalotins with high inhibitory activity towards
Meloidogyne incognita and low activity towards
Caenorhabditis elegans (Mayer et al. 1997, 1999;
Sterner et al. 1997). The nematicidal properties of
the hydroxylated omphalotins are higher than
those of the parent compound, but unfortunately
they are not stable (Büchel et al. 1998a, Liermann
et al. 2009).
Antiviral properties have been reported for
sansalvamide A, a cyclodepsipeptide from a ma-
rine Fusarium, which inhibits viral topoisomer-
ase-catalyzed DNA relaxation (Hwang et al. 1999).
The clavariopsins, cyclic depsipetides from
Clavariopsis aquatica, show selective antifungal
activity, bacteria are not affected and mice tolerate
100 mg/kg of clavariopsin A. As mode of action,
an inhibition of cell components was proposed
(Kaida et al. 2001). Glomosporin from a Glomos-
pora species is a lipophilic depsipeptide with
antifungal activity (Sato et al. 2000). Whether
this compound also inhibits cell wall synthesis
was not reported. Antifungal and cytotoxic activ-
ities were reported for petriellin A (Lee et al.
1995). Cytotoxic activities are exhibited by many
cyclopeptides and -depesipeptides. The destrux-
ins have been intensively investigated (Vey et al.
2002; Skrobek and Butt 2005). Psychrophilin
D is weakly cytotoxic towards P388 mouse
289
leukaemia cells with an IC50 value of 10 mg/ml
(Dalsgaard et al. 2005), while the icosalides inhibit
the replication of MDCK cells with LD50 of 5–10
mg/ml (Boros et al. 2006). The aspergillicins are
weakly cytotoxic with LD99 of 25–50 mg/ml (Capon
et al. 2003).
As inhibitors of 1,3-b-glucan synthesis have
high potential as antimycotic drugs (Fostel and
Lartey 2000), fungi have been intensively screened
for the production of inhibitors of cell wall syn-
thesis and cyclic peptides as well as cyclic depsi-
peptides have been found.
The antimycotic drugs already on the market
(caspofungin, micafungin, anidulafungin) are
derived from lipopeptides (Butler 2004; Morrison
2006). Their spectrum of activity is mainly re-
stricted to Candida and Aspergillus species. Cryp-
tococcus neoformans, Trichosporon and Fusarium
species or Zygomycetes are not affected (Denning
2003), although the glucan synthase from C. neo-
formans is sensitive to echinocandins (Maligie
and Selitrennikoff 2005).
IV. Ecological Role of Cyclic Peptides
and Depsipeptides
Many secondary metabolites play a crucial role for
fungi in their natural habitats. Endophytic fungi
of grasses belonging to the genera Neotyphodium/
Epichloe confer protection from mammalian and
insect herbivores, or enhanced resistance against
nematodes and phytopathogenic fungi (Schardl
et al. 2004; Panaccione et al. 2006). Some of these
beneficial effects are due to NRPS products. Ergo-
valine has been identified among the fungal meta-
bolites in the plant host. Malformins have been
detected in onion scales after infection with
A. niger (Curtis et al. 1974).
The role of siderophores in plant and human
pathogens is currently elucidated by many re-
search groups (for a review see Haas et al. 2008).
Additional functions of siderophores for the pro-
ducing organism are acquisition and storage of
iron as well as regulation of asexual and sexual
development and protection against oxidative
stress (Einsendle et al. 2006; Hof et al. 2009).
Nonproducing organisms like Saccharomyces cer-
evisiae are able to use, e.g. transport iron-side-
rophore complexes, thus the compounds might
also play a role in fungus–fungus interactions.
290 Heidrun Anke and Luis Antelo
In plant-pathogenic fungi cyclic peptides like
HC-toxins in Cochliobolus carbonum, AM toxins in
Alternaria alternata, sirodesmin PL in Lepto-
sphaeria maculans (anamorph Phoma lingam) or
enniatins in Fusarium species act as putative viru-
lence factors. In some cases this has already
been proven, when gene deletions result in apatho-
genic strains or strains with reduced virulence
(Ahn and Walton 1998; Pedley and Walton 2001;
Elliott et al. 2007). Likewise the insecticidal depsi-
petides of insect pathogens have the same function.
Investigation on the role of destruxins in the path-
ogenicity of Metarhizium anisopliae against three
species of insects revealed a direct relationship
between the titer of destruxins produced by the
strains in vitro and their destructive action (Ker-
shaw et al. 1999). In the plant-pathogenic Alter-
naria brassicae, destruxin B is a host-specific
toxin. In three Brassica species the degree of their
sensitivity to destruxin B positively correlated with
their degree of susceptibility (Pedras et al. 2002).
The function of shearamide A, an insecticidal
cyclopeptide isolated from the ascostromata of
Eupenicillium shearii (Belofsky et al. 1998) may
be in protecting the fungus against insects, similar
to ergopeptides in the sklerotia of Claviceps spe-
cies (Chap. 9).
V. Conclusions
The capability to produce secondary metabolites
derived from amino acids by NRPS is widespread
among the higher fungi and not dependent on the
ecological niches inhabited by them. There are no
special habitats from which highly prolific sec-
ondary metabolite producers are isolated.
Cyclic peptides and -depsipeptides constitute
an interesting class of secondary metabolites with
great potential not only in medicine but also in
agriculture. This can easily be grasped from the
wide array of biological activities exhibited by
these compounds. Their chemical diversity is
enhanced by the possibility of producing an
array of related compounds by precursor-supple-
mented fermentations of the correspondent
fungus. This readily facilitates investigations on
structure–activity relationships.
In agriculture, fungally derived pesticides
offer ecological advantages and strains with en-
hanced production of bioactive compounds might
be developed as biopesticides. For both agriculture
and pharmacology bioactive natural compounds
may lead to novel targets and serve as lead
structures.
Acknowledgements. Work in our Institute was supported
by the State of Rhineland–Palatinate, BASF SE, Bayer AG,
BMBF and the DFG.
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14 Fungal Genome Mining and Activation of Silent Gene Clusters

AXEL A. BRAKHAGE1, SEBASTIAN BERGMANN1, JULIA SCHUEMANN2, KIRSTIN SCHERLACH2, VOLKER SCHROECKH1,
CHRISTIAN HERTWECK2

CONTENTS ue to be, a leading source of molecules for drug

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 discovery, but new technologies are required to
II. Fungal Genome Mining and Activation increase the probability of identifying new entities
of Silent Gene Clusters . . . . . . . . . . . . . . . . . . . . . . . . . . 297 (McAlpine et al. 2005).
A. Genetic Potential for Secondary
Metabolism Biosynthesis of Fungi . . . . . . . . . 297
B. Genome Mining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
C. Activation of Silent Gene Clusters . . . . . . . . . 299
III. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 II. Fungal Genome Mining
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
and Activation of Silent
Gene Clusters
I. Introduction A. Genetic Potential for Secondary Metabolism
Biosynthesis of Fungi
The metabolism of all organisms can be divided
into two parts: primary metabolism provides the A literature survey covering more than 23 000
cells with energy and chemical precursors that are bioactive microbial products, i.e. antifungal, anti-
essential for growth and reproduction of the bacterial, antiviral, cytotoxic and immunosup-
organisms; secondary metabolism seems to pos- pressive agents, shows that the producing strains
sess no obvious function for cell growth in the are mainly from the fungal kingdom (ca. 42%),
laboratory (Bennett and Bentley 1989). However, followed by strains belonging to the genus Strep-
these compounds remain a constant source of tomyces (32.1%; Lazzarini et al. 2000). Hence,
drug leads with more than 40% of new chemical fungi represent one of the most promising sources
entities reported since 1981 from microbial of bioactive compounds (Bhatnagar et al. 2002).
sources (Clardy and Walsh 2004; Khosla 1997; Despite the large number of known fungal
Sieber and Marahiel 2005). Even more remarkable metabolites, the biosynthetic potential of these
is the fact that more than 60% of anticancer agents organisms is greatly underestimated by an order
and 70% of anti-infectives currently in clinical use of magnitude. Analyzing the increasing number of
are natural products or natural product-based sequenced genomes indicates that fungi encode
compounds (Yin et al. 2007). Secondary metabo- the genetic information for the biosynthesis
lites from microorganisms have been, and contin- of many more yet unknown compounds (e.g.
Bergmann et al. 2007; Chiang et al. 2008; Peric-
Concha and Long 2003). This information can be
gained bioinformatically by scanning the genome
for the presence of characteristic biosynthesis
genes. Many biologically active compounds are
1
Molecular and Applied Microbiology, Leibniz Institute for Natu- produced by multifunctional enzymes, namely
ral Product Research and Infection Biology (HKI), and Friedrich polyketide synthases (PKSs) and nonribosomal
Schiller University, Beutenbergstrasse 11a, 07745 Jena, Germany;
peptide synthetases (NRPSs). PKSs and NRPSs
e-mail: axel.brakhage@hki-jena.de
2
Biomolecular Chemistry, Leibniz Institute for Natural Product utilize simple malonyl (PKS) or amino acid
Research and Infection Biology (HKI), and Friedrich Schiller (NRPS) building blocks, respectively. They fol-
University, Beutenbergstrasse 11a, 07745 Jena, Germany low very similar strategies for their assembly.

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds)
© Springer‐Verlag Berlin Heidelberg 2009
298 Axel A. Brakhage et al.
Although they utilize different classes of sub-
strates, both PKSs and NRPSs show striking simi-
larities in the architecture and mechanism of the
modularized assembly lines. Each module is re-
sponsible for one or more chain-elongation steps
and can be subdivided into domains controlling
the choice of the extender unit and connection
with the growing peptide chain. A typical NRPS
module minimally consists of an adenylation (A)
domain responsible for amino acid activation, a
condensation (c) domain, connecting the acti-
vated amino acid with the growing chain, and a
thiolation (T) domain – also known as peptidyl
carrier protein (PCP) that serves as an anchor for
the growing peptide chain. Additionally, a variety
of optional [e.g. methyltransferase (MT) or epi-
merisation (E)] domains may be present (Walsh
et al. 2001). Similarly, three domains are the basic
equipment of most PKS elongation modules: an
acyltransferase (AT) domain for extender unit
selection and transfer, an acyl carrier protein
(ACP) for extender unit loading and a ketoacyl
synthase (KS) domain for decarboxylative con-
densation of the extender unit (usually malonyl-
CoA) with an acyl thioester. The resulting b-keto
thioester may subsequently be processed by
b-ketoacyl reductase (KR) domains, dehydratase
(DH) domains, enoyl reductase (ER) domains and
MT domains (Cane et al. 1997). Modular PKS and
NRPS systems can also closely cooperate to form
so-called hybrid products (Cane and Walsh 1999;
Paitan et al. 1999; Silakowski et al. 1999; Wenzel
et al. 2005). Typically, the nascent polyketide or
peptide backbone is further modified by tailoring
enzymes in post-PKS or post-NRPS steps of the
pathway, such as oxygenases and glycosyltrans-
ferases or other transferases, in order to imbue
additional structural functionalities to the final nat-
ural product.
B. Genome Mining
The conserved features within these PKS and
NRPS machineries have been cornerstones of the
genomic-guided discovery of natural products
(Ikeda et al. 2003; Liu et al. 2002; Menzella et al.
2005; Yin et al. 2007). In all cases known to date,
genes codings for PKS and NRPS are part of a
cluster containing additional genes for modifying
enzymes. With the genome sequence at hand, it is
possible to estimate the biosynthetic potential
for a given organism by in silico screening for
typical secondary metabolite biosynthesis genes.
Systematic mining of the genomes has uncovered
the occurrence of cryptic biosynthesis genes in
actinomycetes (McAlpine et al. 2005; Lautru
et al. 2005; Zazopoulos et al. 2003; Challis 2007),
myxobacteria (Wenzel et al. 2005; Bode and Mull-
er 2005), Pseudomonas (Brendel et al. 2007; Gross
et al. 2007) and Burkholderia (Nguyen et al. 2008).
Recently the genomes of various filamentous
fungi, including Aspergillus nidulans,
A. fumigatus and A. oryzae, have been sequenced
(http://www.broad.mit.edu/annotation/genome/
aspergillus_group/MultiHome.html).
The analysis of deduced gene products sug-
gests that A. nidulans has the potential to generate
up to 32 polyketides, 14 nonribosomal peptides
and two indole alkaloids; similar predictions can
be made from the A. fumigatus and A. oryzae
genome data (Table 14.1). Interestingly, there
appear to be almost no orthologs among these
genes across the three species, thus representing
a loss of synteny to a degree not seen in other
regions of the genomes (Bok et al. 2006). This high
number of putative metabolites is greater than the
known metabolites ascribed to these species, and
it may be a reflection of incomplete natural prod-
uct analysis in these species or failure of many
clusters to be expressed, at least under the culture
conditions commonly used in laboratories
(Scherlach and Hertweck 2006). For example, the
aflatoxin gene cluster is not expressed in A. oryzae
(van den Broek et al. 2001; Zhang et al. 2004; Bok
et al. 2006). It is apparent that this vast number of
predicted fungal biosynthesis genes is not
reflected by the metabolic profile observed under
standard fermentation conditions. Apparently, in
the absence of a particular trigger these gene loci
remain silent.
One of the major challenges is to understand
the physiological meaning of the many secondary
metabolites for the producing microorganism
Table 14.1 Putative secondary metabolism gene types.
NRPS Nonribosomal peptide synthetase, PKS polyketide
synthase, DMAT dimethylallyl tryptophan synthase
(Brakhage et al. 2008)
Putative Aspergillus A. fumigatus A. oryzae
gene nidulans
NRPS 14a 14a 17
PKS 32a 14a 28
DMAT 2 7 2
a
Includes one hybrid PKS/NRPS.
 Fungal Genome Mining and Activation of Silent Gene Clusters
E n viro n m e n ta l S ig n a ls
S ig n a l tra n s d u c tio n
C yto p la s m
N u c le a r im p o rt
T ra n s c rip tio n fa c to rs
N u c le u s
S e c o n d a ry
m e ta b o lis m
g e n e c lu s te r
N a tu ra l p ro d u c ts
E ffe c ts ?
(Fig. 14.1). As long as this is not understood it is
often not possible to directly activate gene clusters
under natural conditions and to predict the regu-
latory circuits involved in the regulation of their
biosyntheses. Notably, in many fungal PKS gene
clusters genes encoding transcription factors can
be found, while such genes are scarce in NRPS
gene clusters.
C. Activation of Silent Gene Clusters
For the functional analysis of cryptic or orphan
genes, gene inactivation would only provide an
option if the metabolite was constitutively pro-
duced by the strain. This method is clearly not
applicable for gene clusters that are silent under
standard fermentation conditions, and thus tools
are needed to assign their biosynthetic role. In
general, several strategies would be conceivable:
gene expression could be induced, for example (i)
by heterologous expression under the control of
defined promoters (Fig. 14.2a) or (ii) by promoter
exchange within the genome (homologous expres-
sion; Fig. 14.2b). The latter method would be fea-
sible in particular for bacterial gene clusters with
polycistronic gene organization. In the case of
fungal secondary metabolism gene clusters, pro-
moter exchange can be cumbersome and is usually
employed for individual genes only (Kennedy and
Turner 1996). Obviously the latter approach has
299
Fig. 14.1. Scheme of the pro-
cesses involved in the activation
of fungal secondary metabolism
gene clusters in a fungal cell
numerous disadvantages. The method requires
the availability of genetic techniques for the par-
ticular organism. Furthermore, many fungi only
show a very low frequency of homologous recom-
bination, which hampers promoter exchange. In
addition to the cumbersome handling of large
gene constructs, another drawback is that over-
expression of a single gene of a cluster often leads
to limitation of another gene product of the same
cluster. Finally, gene expression and, as in the
lovastatin pathway, the functioning of enzymes
may be context-dependent (Bergmann et al. 2007).
One solution of this problem is: (iii) the
expression of pathway-specific regulatory genes,
which are present in many secondary metabolite
gene clusters (Fig. 14.2c). This approach is ren-
dered feasible by the fact that all of the genes
encoding the large number of enzymes required
for the synthesis of a typical secondary metabolite
are clustered and that in some cases a single regu-
lator controls the expression of all members of a
gene cluster to a certain extent (McAlpine et al.
2005). Consequently, only a small gene needs to be
handled. Furthermore, ectopic integration of sim-
ple gene cassettes would be sufficient, bypassing
all limitations of homologous recombination.
Most conveniently, this strategy would allow for
the concerted expression of all pathway genes.
Recently, we developed the latter strategy for
the successful induction of a silent metabolic
pathway in the fungal model organism A. nidulans.
300
A PKS/NRPS
PKS/NRPS
: Promoter
PKS/NRPS
TF : Transcription factor
PKS/NRPS
Fig. 14.2. Molecular methods to induce silent gene clusters. (a) Expression of gene clusters in a heterologous host.
(b) Promoter exchange of individual genes of a cluster using strong, regulatable promoters. (c) Overexpression of
transcription factors using strong inducible promoters
By mining the A. nidulans genome for cryptic (or
orphan) gene clusters potentially coding for the
biosynthesis of polyketides or polypeptides, we
noted the presence of a putative hybrid PKS-
NRPS gene (apdA) (Table 14.1, Fig. 14.3). The
deduced gene product shows typical motifs of
PKS and NRPS domains, as well as an additional
C-terminal reductase domain. It obviously repre-
sents a rare fungal PKS-NRPS hybrid. Despite
the large number of metabolic data available for
A. nidulans the role of this gene was obscure. To
date, only a few fungal PKS-NRPS hybrid synthases
have been attributed to metabolic functions. Gene
inactivation experiments revealed that two related
ones are involved in the biosynthesis of the tetra-
mic acid derivatives equisetin and fusarin in Fusar-
ium heterosporum and F. fujikuroi, respectively
(Sims et al. 2005; Song et al. 2004). The cryptic
PKS-NRPS-encoding gene in the A. nidulans
genome is flanked by upstream genes coding for
several putative oxidoreductases: two cytochrome
P450-monooxygenases (ApdB, ApdE), an FAD-
dependent monooxygenase (ApdD) and an enoyl
reductase (ApdC). Downstream of apdA the puta-
tive exporter apdF and activator apdR genes are
Axel A. Brakhage et al.
TF
TF
TF
TF
heterologous host
TF
TF
TF
TF
located. The deduced gene product of apdR is
related to a putative C6 transcription factor of A.
fumigatus and to a putative regulator with a GAL4-
type Zn2Cys6 binuclear cluster DNA-binding do-
main from A. flavus. To prove the concept that the
homologous overexpression of a regulatory gene
can lead to activation of a silent gene cluster the
putative activator gene apdR was amplified from
genomic DNA and cloned into an expression vector
carrying the promoter of the alcohol dehydroge-
nase of A. nidulans (Fig. 14.3). This promoter can
be induced by the addition of cyclopentanone to
the medium and repressed by the use of glucose as
the carbon source (Waring et al. 1989).
Transformants of A. nidulans carrying the
alcAp-apdR gene fusion ectopically integrated
into the genome were checked for transcription
of genes of the cluster by Northern blot analysis.
Whereas the transcripts were completely absent in
the wild type under both non-inducing and induc-
ing conditions, in the transformant strain under
inducing conditions strong mRNA signals were
detected for the genes of the cluster including
the PKS-NRPS hybrid (Fig. 14.4). This observation
further confirmed that under the conditions
 Fungal Genome Mining and Activation of Silent Gene Clusters 301

apd B C D E A F R G

alcAp apdR

KS AT DH MT KR ACP C A PCP RED apdA

ER
*

HO
OH O *
R
* *
Transformant
N O
H Wildtype
R= H: aspyridone A 20 22 24 26 28
R= OH: aspyridone B time [min]

Fig. 14.3. Activation of a silent gene cluster in Aspergillus nidulans by overexpression of the transcription factor gene
apdR using the inducible promoter of the alcohol dehydrogenase gene alcA of A. nidulans. This leads to the production of
a mixed PKS-NRPS encoded by the apdA gene and the production of the novel natural products aspyridone A and B
under inducing conditions which is shown by the HPLC profile (Bergmann et al. 2007). New metabolites are labeled by
asterisks

apdB apdC apdD apdE

wt transformant wt transformant wt transformant wt transformant
R I R I R I R I R I R I R I R I

apdA apdF apdR apdG

wt transformant wt transformant wt transformant wt transformant
R I R I R I R I R I R I R I R I

Fig. 14.4. Northern blot analysis demonstrating the activation of the silent gene cluster under inducing conditions in the
transformant strain encoding the extra copies of the inducible trancription factor apdR. R repressing conditions;
I inducing conditions with cyclopentanone in the medium; apdR transcription factor gene (Bergmann et al. 2007)

applied this gene cluster was silent without induc- inducing conditions, the transformant strain pro-
tion. Furthermore, it also helped to confine the duced novel compounds called aspyridones
borders of the gene cluster (Bergmann et al. 2007). (Fig. 14.3). They are similar, but not identical
The culture extracts were analysed by to a variety of other pyridones isolated from
HPLC coupled to DAD and MS detectors. Under fungi, such as militarinone D and tenellin
302 Axel A. Brakhage et al.
(Eley et al. 2007; Schmidt et al. 2003). In a broad
bioactivity screening they exhibited moderate
cytotoxic activities. The results cited here provide
the proof of principle for a strategy that may be
generally applicable to the activation of silent bio-
synthesis gene clusters, in particular in eukaryotes.
Recently, rather global regulatory strategies
were explored for the activation of silent fungal
biosynthesis genes. It is known that some second-
ary metabolite gene clusters are located in chroma-
tin regions that are controlled by epigenetic
regulation such as histone deacetylation and DNA
methylation. Keller and colleagues have demon-
strated that the disruption of histone deacetylase
activity (Dhda) in A. nidulans led to transcriptional
activation of secondary metabolite gene clusters
(Perrin et al. 2007; Shwab et al. 2007). This obser-
vation stimulated the use of epigenetic modifiers
for inducing silent natural product biosynthetic
pathways. Cichewicz and colleagues successfully
applied this concept for remodeling the metabo-
lome in various filamentous fungi (Williams et al.
2008).
III. Conclusions
The rapidly growing number of sequenced
fungal genomes reveals that the biosynthetic pot-
ential of fungi has been greatly under-explored by
traditional methods of natural product discovery.
Obviously, a multitude of potentially useful meta-
bolites still awaits discovery. Molecular methods
indicate that many gene clusters are silent under
standard laboratory cultivation conditions. How-
ever, if these genes are still intact, their expression
by genetic engineering may lead to the discovery
of so far unknown natural products and therefore
potential drug candidates.
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15 Non-Ribosomal Peptide Synthetases of Fungi

KATRIN EISFELD1

CONTENTS Myxobacteria) and filamentous fungi. Secondary

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 metabolites are compounds that are not directly
II. Non-Ribosomal Peptide Synthetases . . . . . . . . . . 306 involved in processes indispensable to life but
A. Structure of NRPSs . . . . . . . . . . . . . . . . . . . . . . . . . 306 often have potent physiological activities. Al-
B. Module Arrangement . . . . . . . . . . . . . . . . . . . . . . . 307 though chemically diverse, all secondary metabo-
C. Domain Types of Non-Ribosomal
Peptide Synthetases . . . . . . . . . . . . . . . . . . . . . . . . . 309 lites are produced by a few common biosynthetic
1. Adenylation Domain . . . . . . . . . . . . . . . . . . . 309 pathways, including non-ribosomal peptide syn-
a) Functions of Adenylation thesis (Keller et al. 2005).
Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Fungal and bacterial NRPs are a structurally
b) Substrate Specificity . . . . . . . . . . . . . . 310 very diverse family of natural products which dis-
2. Thiolation Domains and
4’-Phosphopantetheine play a wide variety of biological activities. The most
Transferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 famous non-ribosomally produced peptide is peni-
3. Condensation Domains . . . . . . . . . . . . . . . . 313 cillin which was discovered by Alexander Fleming’s
4. Modifying Domains . . . . . . . . . . . . . . . . . . . . 313 observation on the inhibition of bacterial growth
a) N-Methylation Domain . . . . . . . . . . . . 314 by Penicillium notatum (Fleming 1929). This anti-
b) Epimerization Domains and
Amino Acid Racemases . . . . . . . . . . 314 biotic gained importance during World War II
5. Termination Domains . . . . . . . . . . . . . . . . . . 315 when penicillin was used as a “miracle drug” and
a) Thioesterase Domain . . . . . . . . . . . . . 315 saved countless lives. Hence many NRPs (e.g. pen-
b) Reductase Domain . . . . . . . . . . . . . . . . 315 icillin, cyclosporin, ergotamine) have tremendous
c) Condensation Domain . . . . . . . . . . . . 315 importance as pharmaceutically relevant agents.
III. Distinctions Between Fungal and Bacterial
NRPSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Many peptides with structures unique to fungi are
IV. Non-Ribosomal Peptide Synthesis known, including siderophores of the ferrichrome
in Basidiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 and coprogen type, cyclodepsipeptides and peptai-
V. Physiological Significance of Peptides . . . . . . . . . 318 bols. Biosynthesis of these compounds is indepen-
VI. Examples for NRPS Gene Clusters dent of ribosomes and mRNA but is catalyzed by
and Cluster Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
A. Penicillin and Cephalosporin Biosynthesis . . 320 large multifunctional enzymes called non-ribosom-
B. Ergot Alkaloid Biosynthetic Gene Clusters . . 321 al peptide synthetases (NRPSs) which are discussed
VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 here. NRPSs are the largest enzymes known in
A. Approaches to Identify New NRPSs . . . . . . . 323 nature. Peptides produced by NRPSs show peculiar
B. Combinatorial Biosynthesis of New NRPs . . 324 features compared to traditional proteins. They
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
contain not only standard amino acids but also
non-proteinogenic amino acids. Peptides can be
linear, but there also exist cyclic and bran-
I. Introduction ched NRPS configurations. The amino acids
incorporated can be modified by N-methylation,
Non-ribosomal peptides (NRPs) are a class of epimerization or can be reduced. Sizes of the
secondary metabolites produced by bacteria non-ribosomally produced peptides range from
(Bacilli, Streptomyces, some Pseudomonades and two to 40 amino acids (for reviews see for example
Stachelhaus and Marahiel 1995; Kleinkauf and von
Döhren 1996; Schwarzer and Marahiel 2001;
1
Institute of Biotechnology and Drug Research, Erwin-Schrödin- Schwarzer et al. 2003; Finking and Marahiel 2004;
ger-Strasse 56, 67663Kaiserslautern, Germany;
e-mail: eisfeld@ibwf.de
von Döhren 2004).

Physiology and Genetics, Ist Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
306 Katrin Eisfeld
Protein data show that NRPSs exhibit a mod-
ular structure, with one module being a semi-
autonomous unit that recognizes, activates and
modifies a single residue of the final peptide. The
modules themselves are organized in domains
with specific catalytic functions, thereby defining
the amino acid sequence and structure of the
products (Smith et al. 1990; Zocher and Keller
1997; Konz and Marahiel 1999). Some non-ribo-
somal peptide synthetases (including enniatin
synthetase, PF1022 synthetase, beavericin synthe-
tase) have been purified and enzymatically char-
acterized (Zocher et al. 1982; Peeters et al. 1988;
Weckwerth et al. 2000). Biochemical investiga-
tions reveal that these enzymes consist of one
single polypeptide chain which comprises all cat-
alytic functions necessary for peptide synthesis,
i.e. ATP-dependent substrate activation, thiola-
tion reactions, N-methylation and condensation
of the covalently bound substrates. To date, far
more peptides which are supposed to be produced
non-ribosomally are known than the genes that
encode their corresponding NRPSs. Table 15.1
gives a summary of NRPs from ascomycetes
whose biosynthetic pathways have been fully
characterized (fungal siderophore synthetases
are not included; they were reviewed very recently
by Johnson 2008).
In genome sequencing projects a great num-
ber of new NRPS genes have been identified while
the products of the NRPSs they encode have not
been detected. For example, in Aspergillus fumi-
gatus a total of at least 14 NRPS genes have been
discovered, but only four of them can be asso-
ciated with their NRPS products: the gliotoxin
synthetase gene (gliP), the ferricrocin synthetase
gene (sidC), the gene encoding fusarinine synthe-
tase (sidD) and the brevanamide synthetase gene
ftmA (Nierman et al. 2005; Reiber et al., 2005;
Balibar and Walsh 2006; Cramer et al. 2006;
Maiya et al. 2006; Schrettl et al. 2007; Stack et al.
2007). Other fungi of the genus Aspergillus also
produce a considerable variety of non-riboso-
mally produced peptides. However, although
conserved orthologous NRPS genes exist, the
products of most of the NRPSs remain unknown.
In A. terreus for example, only four of a total of 20
NRPS genes can be assigned to a hypothetical
function by means of homology comparison
(Cramer et al. 2006). Cross-genome comparisons
indicate that most filamentous ascomycetes
carry many genes encoding NRPSs. Some of
them appear to be discontinuously distributed,
while most are not conserved from one species to
another (Turgeon et al. 2008). This implies that a
lot of unknown NRPS products exhibiting poten-
tially interesting properties still await discovery.
II. Non-Ribosomal Peptide Synthetases
A. Structure of NRPSs
NRPSs have been proposed to operate via a
thiotemplate mechanism with a pantotheine
“swinging arm” transferring activated amino
acids to the condensation domain of the enzyme
(Stein et al. 1996). A multimodular arrangement of
NRPSs was confirmed by the isolation and charac-
terization of genes encoding NRPSs from bacterial
and fungal origin. One module is responsible for
the introduction of one amino acid. Each module
consists of several domains with defined functions
(Stachelhaus and Marahiel 1995; Marahiel 1997;
Schwarzer and Marahiel 2001; Weber and Marahiel
2001; Mootz et al. 2002a, b). Usually the order of
modules and domains of a fungal NRPS is collinear
to the sequence of the synthesized product. Unlike
in bacteria, fungal non-ribosomal peptide synthesis
is mostly catalyzed by one enzyme. These enzymes
can reach an enormous size; for example, the Tri-
choderma virens PES gene which is responsible for
peptaibol production comprises a 62.8-kb continu-
ous open reading frame and the peptide synthetase
consists of 18 modules (Wiest et al. 2002).
A minimal module for incorporation of one
amino acid into the growing peptide chain con-
sists of an adenylation (A) domain, a thiolation
(T) or peptidyl carrier protein (PCP) domain
and a condensation (C) domain. The A domain
is responsible for substrate recognition and
activation, the T/PCP domain binds a 40 -phospho-
pantetheine cofactor to which the activated amino
acid is tethered and the C domain is required for
peptide bond formation. Besides the domains
being essential for the elongation of the peptide
chain there are optional domains including
domains for N-methylation (N-MTase domain),
epimerization (E domain) and heterocyclic ring
formation which are inserted at specific locations
within the module. Furthermore, besides protei-
nogenic amino acids often unusual amino acids
or carboxylic acids are incorporated. Further
variability is achieved through cyclization of the
 Non-Ribosomal Peptide Synthetases of Fungi 307

Table 15.1. Summary of characterized NRPSs, their products and properties

Producer NRPS/Structure Product Properties

Penicillium ACV synthetase (ACVS) Tripeptide Precursor of penicillin and
chrysogenum (ATC)2ATETe cephalosporin
and others
Fusarium Enniatin synthetase Cyclo-hexadepsipeptide Ionophore; inhibitor of acyl-
oxysporum and CATCAMTTC CoA-cholesterol
others acyltransferase and of
phosphodiesterase; potential
anticancer compound.
Beauveria nivea, Cyclosporin synthetase 11-Cyclopeptide Immunosuppressive: antifungal;
Tolypocladium CAT(CAMT)4CAT anti-inflammatory
niveum (CAMT)2CATCAMTCATC
Claviceps purpurea Ergotamin synthetases Acyl-tripeptides Aeurotoxic;
ATC and ATCATCATCyc vasoconstrictor
Cochliobolus HC-toxin synthetase (HTS1) Cyclo-tetrapeptide Inhibitor of histone-
carbonum ATEC(ATC)3 deacetylases: maize-
pathogen
Alternaria alternata AM-toxin synthetase(ATC)4 4-Peptidolactone Phytotoxic
Trichoderma virens Peptaibol synthetase Acylpeptides (18, 14 or 11 fungicidal
KSa-ACb-C(ATC)17 AT-DHc amino acids)
Leptosphaeria Sirodesmin synthetase SirP Diketopiperazine Phytotoxin mycotoxin;
maculans (ATC)2 epipolythiodioxopiperazin antibacterial activity
Aspergillus Gliotoxin synthetase(ATC)2T Cyclodipeptide Induces apoptosis;
fumigatus and immunosuppressive
others
A. fumigatus Brevianamide F synthetase Diketopiperazine Precursor of fumitremorgins
(ATC)2
Epichloe festucae Peramine synthetase perA Pyrrolopyrazine Repellent to insects
ATCAMTR
A. nidulans TdiA Bisindolebenzoquinone Precursor of terrequinone
ATTe
Xylaria sp. BCC1067 Bassianolide synthetase Cyclooctadepsipeptide Pathogenic to insects
CATCAMTTCR
a
KS = Ketoacyl synthase.
b
AC = Acyltransferase.
c
DH = b-Hydroxysteroid dehydrogenase.

peptide or modifications that take place after the
assembly of the peptide chain (Stachelhaus and
Marahiel 1995; Marahiel 1997; Schwarzer and
Marahiel 2001; Weber and Marahiel 2001; Mootz
et al. 2002a, b; Samel and Marahiel 2008).
Two types of modules can be distinguished:
the initiation or starting module lacks the C do-
main and therefore is A–T. Elongation or extend-
ing modules are –C–A–T–. Methylation domains
are located within A-domains, while epimeriza-
tion domains are inserted between T– and
C-domains. The products are released at thioes-
terase-like domains, reductive domains or
specialized C-domains. Fig. 15.1 shows the do-
main composition within one module.
Comparison of bacterial and fungal NRPSs led
to the identification of conserved sequence motifs
(“core motifs”) characteristic for each domain
type (Stachelhaus and Marahiel 1995; Konz and
Marahiel 1999). These motifs are similar in bacte-
ria and fungi but differ to some extend. Table 15.2
displays a comparison of bacterial and fungal
NRPS core motifs.
B. Module Arrangement
Originally, many linearly organized NRPS
enzymes were discovered. This led to the develop-
ment of the co-linearity rule: the order in which
the amino or hydroxyl acids are combined into
the peptide product is normally determined by the
arrangement of the A domains within the multi-
domain NRPS enzyme. However, in the meantime
many NRPSs with alternative synthesis patterns
308 Katrin Eisfeld

gene
1 2

modules
A adenylation domain

domains C condensation domain

A T C A T
SH SH T thiolation domain

Fig. 15.1. The amino acid sequence of NRPSs, deriving
from the gene, allows identification of the modules
responsible for amino acid activation. Modules can be
subdivided into domains. Adenylation (A) domains
are responsible for substrate recognition and activation,
thiolation (T ) domains covalentely bind the activated
substrate and the condensation (C) domains are the
site for peptide bond formation. Modules lacking a
C domain are used for initiation of peptide synthesis,
while those harboring a C domain are elongation modules
(from Finking and Marahiel 2004)

Table 15.2. Conserved sequence motifs of NRPS domains

Domain Motif Sequence in bacteria Sequence in C. heterostrophus Sequence in O. olearius

(Stachelhaus and Marahiel (ascomycete; Lee et al. 2005) (basidiomycete; Eisfeld,
1995) unpublished results)
A A1 L(TS)YxEL LTYxEL xTYxxL
A2 LKAGxAYL(VL)P(LI)D LKAGA(AG)F(VY)P(LI)DP LKAGxAxxPxx
A3 LAYxxYTSG(ST)TGxPGK LAY(VI)(IL)FTSGTGxPKGV xAYxxxTSG(TS)TGxPKxV
A4 FDxS FDxSxxE FDxxxx(ED)
A5 NxYGPTE N(GA)YGP(TA)E NxYGPxE
A6 GELxIxGxG(VL)ARGYL GELxIxxxGxG(VL)ARGYL GELx(IVL)GGxxx(AG)xGYxx
A7 Y(RK)TGDL RxY(RK)TGDLxR X(YF)xTGDxxR
A8 GRxDxQVKIRGxRIELGEIE GRxDxQVK(IL)RGxR(IV)ELGEIE GRxDxxxVKxxGxR(VI)xLxE
(IV)x
A9 LPxYM(IV)P LPxYM(IV)P LPxxMxP
A10 NGK(VL)DR SGKxDR xGKxDR
C C1 Not listed PxxPxQ PxxPxQ
C2 RHExLRTxF xxxxLRTxx xxxxLR(TS)xF
C3 MHHxISDG(WV)S xHHxxxDGxS xHHA(IL)YDGxS
C4 YxD)FY)AVW xxxxAAWA /
C5 (IV)GxFVNT(QL)xR xGPxxxTxPxR xGPxxxTxPVRV
E E1 xxxPIQxWF PLxPIQxxF PLxxxQxxx
E2 HHxISDG(WV)Sx HHLxVDxVSW HHLxxDxVSx
E3 DxLLxAxG Not listed xxLxxxG
E4 EGHGRE Not listed ExHGRx
E5 RTVGWFTxxYP(YV)PF xTVGWFTMxPxxx xTVGWFTxxxPx
E6 PxxGxGYG Not listed PxxGxxYx
E7 FNYLG(QR) Not listed FNxLGx

and hence different strategies of biosynthesis have
been identified. Mootz et al. (2002a, b) proposed
classifying the known NRPS systems into three
groups according to their biosynthetic logic: linear
(type A), iterative (type B) and non-linear NRPSs
(type C). Examples for the three groups can be
found not only in bacterial systems but also in fungi.
A typical linear NRPS template has the do-
main organization A–T–(C–A–T)n1–TE/C with
n being the number of amino acids that form the
peptide. Modification domains can be inserted
into the respective modules. In linear NRPSs, pre-
dictions can be made about the products being
synthesized. Examples for fungal linear NRPS are
 Non-Ribosomal Peptide Synthetases of Fungi
A T C A T C A T C A T C
AM toxin synthetase
HC toxin synthetase A T E C A T C A T C A T C
Enniatin synthetase C A T C A M TT C
SidC A T C A T C A T C T C T C
Pes1 A T E C A C A C A T E C T C T
Pso3 C A T A T C A T E C A T E
ACV synthetase, peptaibol synthetases, and cyclo-
sporin synthetases (Fig. 15.2).
Iterative NRPSs use their modules or domains
repeatedly during the assembly of a product.
For example, in enniatin synthesis, the cyclo-hex-
adepsipeptide is synthesized by repeated use of two
modules which activate D-hydroxy-isovaleric acid
and N-methylvaline (Glinski et al. 2002). PF1022A
synthetase most likely exhibits the same domain
architecture as enniatin synthetase and also pro-
duces a cyclo-hexadepsipeptide which is assembled
by successive condensations of dipeptidol building
blocks (Weckwerth et al. 2000). Fungal siderophore
synthetases of the ferrichrome type combine three
small amino acids (Gly, Ala or Ser) and three acy-
lated L-5-hydroxy-ornithines but contain only three
A domains. These hexapeptides are also expected to
be produced iteratively, mediated by additional
truncated modules lacking the adenylation
domains (Haas et al. 2003; Schwecke et al. 2006).
This architecture is also found at the C-terminus of
the enniatin synthetase Glinski et al. 2002). Mootz
et al. (2002a, b) suggested that iterative NRPSs can
be regarded as linear NRPS with an iterative activity
at the C-terminus (Fig. 15.2).
The architecture of non-linear NRPSs differs
from that of type A NRPSs and the domain
interaction is more complex. Their possible pro-
ducts can not be predicted. An unusual domain
arrangement is found at a large number of bacte-
rial NRPSs while most fungal NRPSs described so
far are of the linear or iterative type.
For example, in syringomycin and yersiniabactin biosynthe-
sis occurs in trans aminoacylation. In yersiniabactin synthe-
sis one A domain loads three different PCPs (Zhang et al.
309
Fig. 15.2. Examples for linear,
iterative and non-linear NRPSs
linear
in fungi
iterative
non-linear
1995; Guenzi et al. 1998; Stachelhaus et al. 1999). Another
example for a type C NRPS is myxochelin synthesis where
one C domain catalyses the formation of two amide bonds
(Silakowski et al. 2000). Non-linear NRPSs are also often
found to incorporate free soluble molecules without prior
binding to a thioester, as in vibriobactin or bleomycin
synthesis (Butterton et al. 2000; Keating et al. 2000a, b;
Wyckoff et al. 2001). By now, in Ascomycetes only three
NRPSs belonging to type C are described: the A. fumigatus
NRPS Afpes1 has the domain organization ATC-CA-ATC-A
(Neville et al. 2005). The Alternaria brassicae NRPS
Abrepsy1 consists of five complete modules and an initia-
tion lacking the A domain (TEC-ATC-ATEC-ATC-ATEC-
TC), similar to yersiniabactin synthetase. Therefore Guil-
lemette et al. (2004) suggested the presence of a second
NRPS with at least one A domain. Stand-alone adenylation
domains can be found performing Blast searches in many
fungal genomes (Eisfeld, unpublished data). Also, the basid-
iomycete O. olearius NRPS Pso3 has an unusual domain
arrangement (CAT-ATC-ATEC-ATE; Eisfeld et al., unpub-
lished data). Unfortunately, the products of these fungal
non-linear NRPSs have not been identified so far. Thus, no
statement about the strategy of synthesis can be made.
C. Domain Types of Non-Ribosomal Peptide
Synthetases
1. Adenylation Domain
a) Functions of Adenylation Domains
The core element of NRPS modules is the adeny-
lation domain (A-domain). This domain is about
550 amino acids in size and fulfils two functions:
the selection and the activation of the cognate
substrate. The substrate to be activated is an
amino acid, an imino acid, a hydroxyl acid or a
carboxy acid that will subsequently be added to
the growing peptide chain (von Döhren 2004). The
activation step is ATP dependent, consuming one
310
ATP and generating one PPi (Dieckmann et al.
1995; Stachelhaus and Marahiel 1995; Mootz and
Marahiel 1997). It has been studied by the ATP-
PPi exchange assay which was developed to mea-
sure the substrate specificity for an A domain
(Santi et al. 1974; Konz et al. 1999; Mootz and
Marahiel 2002).
Adenylation domains catalyze a two-step re-
action. The first step is the adenylation of
the substrate resulting in the formation of an
acyl-AMP. The second step is the formation of
a covalent binding via a thioester bond to the
40 phosphopantetheine (Ppant) cofactor at the
PCP domain (Fig. 15.3).
A set of conserved sequence motifs for recog-
nition and binding of the substrates is character-
istic for A domains (Fig. 15.4). These motifs are
highly conserved and almost all of them are locat-
ed around the active sites where the substrates are
bound (Marahiel et al. 1997).
The activation reaction is analogous to the first reaction
performed by aminoacyl-tRNA synthetases on ribosomes
(Arnez and Moras 1997). Crystal structures exist for two
bacterial adenylation domains, PheA (Conti et al. 1997)
and DhbE (May et al. 2002). PheA is the L-phenylalanine-
activating domain of the Bacillus brevis gramicidin S syn-
thetase GrsA. Conti et al. (1997) found that this A-domain
shares no homology with tRNA synthetases but shows low
(16%) sequence homology with acyl-CoA ligases and fire-
fly luciferases, which also belong to the superfamily of
peptide synthetases and adenylate-forming enzymes and
these enzymes exhibit an almost identical fold. May et al.
(2002) solved the structure of the stand-alone 2,3-dihy-
O O
+ Mg2+ +
H3N H3N
O + ATP
-PPi R
R
Fig. 15.3. The adenylation domain catalyzes the activation
of a carboxy acid by hydrolysis of ATP
A1 A2 A3 A4 A5 A6 A7 A8
A3 interaction with pyrophosphate leaving group
A4 interaction with amino group
A7 interaction via hydrogen bonds with oxygen atoms of nucleoside ribose moiety
A8 essential for adenylation
A10 binds to substrate and ribose oxygens
Fig. 15.4. Position and function of conserved motifs of the adenylation domain
Katrin Eisfeld
droxybenzoic acid activating domain DhbE which sup-
ported the findings that A domains share a very similar
fold. Both adenylation domains consist of two major sub-
domains, a smaller C-terminal subdomain of about 100
residues and a larger N-terminal subdomain of about 400
residues.
b) Substrate Specificity
The study of the specificity of A domains was
greatly facilitated by the determination of the
crystal structures of PheA and DhbE (Conti et al.
1997; May et al. 2002). Based on the crystal struc-
ture of PheA, Conti et al. (1997) determined the
amino acid residues that are crucial for substrate
binding. Adenylation domain sequence align-
ments identified ten sequence positions which lie
in a 100 amino acid stretch between core motifs
A4 and A5 and are lining the binding pocket
(Fig. 15.4). This allowed the correlation with
known substrates and the proposal of a non ribo-
somal specificity-conferring code. The crystal
structure of DhbE allowed the adaptation of a
specificity conferring code for aryl acid activating
domains (Stachelhaus et al. 1999). Challis et al.
(2000) presented a more detailed analysis allowing
the prediction of the specificity of adenylation
domains of unknown function. The predictive
structure-based models permit the prediction of
amino acid specificity in NRPS of unknown func-
tion but derive largely by systematic comparative
analysis of bacterial A domains. However, these
models are not fully applicable for all fungal A
domains. For example, while the three adenylation
domains of ACV synthetases have identical
amino acid residues at the positions defining the
non-ribosomal code, no other valine specificity
domains correlate. The D-alanine-specific
O-AMP
domains of cyclosporin synthetase and HC-toxin
synthetase have identical residues, but the pre-
dicted specificity-conferring residues for alanine
and proline show wide variations in fungi (von
A9 A10
 Non-Ribosomal Peptide Synthetases of Fungi
Döhren 2004). Siderophore synthetases of the fer-
richrome type are restricted to fungi and Schwecke
et al. (2006) analyzed the codons derived from code
positions in detail, as they did not match available
specificities. The authors found three different
codes for glycine activating domains and three
major groups of adenylation domains for orni-
thine-derived substrates. Rausch et al. (2005)
invented a new support vector machine-based ap-
proach to predict substrate specificity using 1230
NRPS A domains and were able to predict the
specificity for 18% more sequences than Stachel-
haus et al. (1999) and Challis et al. (2000). For 2.4%
of the sequences no prognosis was possible, a large
ratio of them of fungal origin. Therefore, eukaryot-
ic A domains might have developed a differing
domain architecture of the substrate binding sites
or an alternative selection of the residues lining the
binding pocket might define slightly different con-
tacts (Rausch et al. 2005; Schwecke et al. 2006).
Furthermore, in contrast to aminoacyl-tRNA
synthetases, A domains of peptide synthetases
often do not display unique substrate specificity.
Many non-ribosomally formed peptides display
both highly conserved and variable amino acid
positions. Wiest et al. (2002) suggested that the
multiplicity of products of one NRPS is a result of
the ability of the A domain to bind multiple sub-
strates. Both in phylogenetic analysis and using the
signature sequences proposed by Challis et al.
(2000) the A domains of peptaibol synthetases
from Trichoderma/Hypocrea display a high diversi-
ty and prediction of amino acids bound by
these modules often was impossible (Komon-
Zelazowska et al. 2007). These variations lead to a
wide range of peptide families. For example, more
than 30 analogues of cyclosporin A have been de-
scribed (Billich and Zocher 1987; Lawen et al. 1989;
Traber et al. 1989; Kleinkauf and von Döhren 1995;
Eckstein and Fung 2003; Wei et al. 2004). Cyclo-
sporin A is a cyclic undecapeptide with various
biological properties including antifungal, antipar-
asitic, anti-inflammatory and immunosuppressive
activities (Borel 1986) and is widely used in human
transplantation surgery (Kahan 1984) and in the
treatment of autoimmune diseases (Talal 1988).
The cyclosporin synthetase of Beauveria nivea
exhibits a broad spectrum of activities. Incorpora-
tion of amino acids ranges from highly specific sites
(e.g. sarcosine at position 3, methylleucine at posi-
tions 4, 6, 9) to very unspecific ones (e.g. butenyl-
methythreonine at position 1, L-a-aminobutyric
311
acid at position 2, alanine at position 8). Interest-
ingly, the specificity of the cyclosporin synthetase
system differs in vivo and in vitro (Lawen and
Traber 1993). Most sites of the related peptolide
SDZ 214-103 synthetase appear to be more specific
than those of cyclosporine synthetase (Lawen and
Traber 1993).
Isolated A domains of ACV synthetase which synthesizes
the penicillin precursor do not discriminate between L-a-
amino-adipic acid (Aad) and valine. In didomain con-
structs with the cognate carrier proteins however, both
substrates were activated at similar levels but only Aad
was detected as an acid-stable thioester, which suggests a
role in selection of the carrier domain (von Döhren et al.
1999). There also exist various natural enniatins caused by
a lowered substrate specificity of the A domain of the
second module (EB) of the enniatin synthetase which
activates the branched chain N-methyl-L-amino acid
(Madry et al. 1984). The first module of the enniatin
synthetase (EA; activation of a-D-hydroxyisovaleric acid)
also has a relaxed substrate specificity (Krause et al. 2001;
Feifel et al. 2007), but a highly specific dehydrogenase
supplies the enzyme exclusively with D-Hiv (Lee et al.
1992) and is found in most enniatin synthesizing strains.
Therefore a high conservation in this position is preserved.
Feifel et al. (2007) demonstrated, that the enniatin synthe-
tase has substrate tolerance towards a variety of different a-
D-hydroxy acids and furthermore permits the synthesis of
various new enniatins with versatile functional groups.
Peptaibols are synthesized by various fungi, includ-
ing fungi of the genera Acremonium, Paecilomyces,
Emericellopsis and several species of Trichoderma
(reviewed by Daniel and Filho 2007). Peptaibols are
linear oligopeptides which self-assemble into oligo-
mers forming pores or channels across the plasma
membrane of their target organisms (Chugh and
Wallace 2001). They typically occur as mixtures of
isoforms and more than 300 peptaibols have been
found to date (Whitmore and Wallace 2004; http://
www.cryst.bbk.ac.uk/peptaibol). A special charac-
teristic of peptaibols is that they are produced as
families of similar peptides, often differing in one
or a few amino acids and displaying similar
biological activities. Particularly noticeable, howev-
er, is that most positions are conserved while
others are less stringently controlled. Two exam-
ples are shown in Fig. 15.5.
2. Thiolation Domains and 40 -
Phosphopantetheine Transferases
Following selection and activation, the substrate is
covalently attached to the 80-amino-acid thiola-
tion (T) or peptidyl carrier protein (PCP) domain
312 Katrin Eisfeld

Antiamoebins (Lehr et al. 2006)

I Ac-Phe-Aib-Aib-Aib-D-Iva-Gly-Leu-Aib-Aib-Hyp-Gln-D-Iva-Hyp-Aib-Pro-Phe-OH
II Ac-Phe-Aib-Aib-Aib-Aib-Gly-Leu-Aib-Aib-Hyp-Gln-D-Iva-Hyp-Aib-Pro-Phe-OH
XVI Ac-Leu-Aib-Aib-Aib-D-Iva-Gly-Leu-Aib-Aib-Hyp-Gln-D-Iva-Hyp-Aib-Pro-Phe-OH

Ampullosporin A and analogues (Kronen et al. 2001)

A Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Gln-Leu-Aib-Gln-Leu-OH
B Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Ala-Aib-Gln-Leu-Aib-Gln-Leu-OH
C Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Aib-Ala-Gln-Leu-Aib-Gln-Leu-OH
D Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Gln-Leu-Ala-Gln-Leu-OH
E1 Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Ala-Ala-Gln-Leu-Aib-Gln-Leu-OH
E2 Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Ala-Ala-Gln-Leu-Ala-Gln-Leu-OH
E3 Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Aib-Aib-Ala-Gln-Leu-Ala-Gln-Leu-OH
E4 Ac-Trp-Ala-Aib-Aib-Leu-Aib-Gln-Ala-Ala-Aib-Gln-Leu-Aib-Gln-Leu-OH

Fig. 15.5. Examples for similar peptides of the peptaibol family. Less conserved positions are shaded in gray

O O In Aspergillus fumigatus and in A. nidulans, the

H3N+ H3N+
O-AMP + SH S
-AMP
R R
T peptides (Kim et al. 2001; Mootz et al. 2002a, b;
Fig. 15.6. Aminoacyl adenylate substrates of the elongat-
ing peptide chain are covalently attached to the 40 Ppant
cofactor of the T domain
located at the C terminus of an A domain. This
domain accepts the activated amino acid and acts
as a transport vehicle (Stachelhaus et al. 1996;
Weber et al. 2000; Fig. 15.6). Before this occurs,
the T domain has to be post-translationally con-
verted to the active form by the covalent attach-
ment of a 4’ Ppant cofactor derived from
coenzyme A (Lambalot et al. 1996; Reuter et al.
1999). This modification is catalyzed by a member
of a special class of phosphopantetheinyl trans-
ferases (the carrier protein superfamily) which
also activate acyl carrier proteins of fatty acid
synthases and polyketide synthases.
Bacterial Ppant transferases (PPTases) are subdivided into
three groups according to their primary sequences and
their substrate specificities (Lambalot et al. 1996). The bac-
terial AcpS-type PPTases are usually associated with pri-
mary metabolism while a second group of eukaryotic
PPTases are integral domains of fatty acid synthases.
Ppant transferases of the third class (sfp type; surfactin
phosphotransferase) often are associated with NRPS gene
clusters (Nakano et al. 1992; Lambalot 1996). These enzy-
mes modify a variety of carrier proteins and domains in-
cluding PCPs of NRPSs and acyl-carrier proteins (ACPs) of
fatty acid synthases and polyketide synthases in bacteria.
npgA/cwfA PPTases have been characterized and
shown to contribute to penicillin biosynthesis,
pigmentation, lysine and siderophore biosynthe-
sis and biosynthesis of other polyketides and
Keszenman-Pereyra et al. 2003; Oberegger et al.
2003; Márquez-Fernández et al. 2007). It was sug-
gested that a single PPTase is responsible for the
activation of all PKSs and NRPSs in A. nidulans
(Márquez-Fernández et al. 2007).
T domains exhibit a conserved structure consisting of a
four-helix bundle fold (Weber et al. 2000). Helix 2 contains
the site of recognition for other protein partners (includ-
ing AcpS, A-domains, C-domains, E-domains; reviewed by
Finking and Marahiel 2004).
In T domains of NRPSs the core motif is LGG
(HD)S(LI). The motif encloses a highly conserved
serine residue whose hydroxyl group functions as
attachment site for Ppant. The activated amino
acid is transferred onto the thiol moiety of Ppant
and a thioester bond is formed. The cofactor acts
according to the swinging arm mechanism de-
scribed for fatty acid synthesis to allow the
bound substrate to travel between different cata-
lytic centers (Finking and Marahiel, 2004; Stachel-
haus et al. 1996; Stein et al. 1996).
Alignments of carrier domains of NRPSs led
to clustering that correlates with function (i.e.
interaction with their protein partners and accept-
ed amino acid; Dieckmann and von Döhren 1997)
and localization of the T domain within a module
(Linne et al. 2001). In bacteria, thiolation domains
interacting with epimerization domains contain
 Non-Ribosomal Peptide Synthetases of Fungi
O O
R1
+ –H2O
H3N H2N
S S SH H3N
+ groups of activated substrates
R1 R2
T T T T
the conserved motif LGGDSL, while interaction
with condensation domains requires the motif
LGGDSI. Again, the assumption that an aspara-
gine residue is essential for the epimerization re-
action is not fully applicable to fungi, as fungal T
domains contain most frequently an isoleucine
residue in the presence and in the absence of an
E domain (von Döhren 2004).
3. Condensation Domains
The condensation (C) domain consists of 450
amino acids and is the third domain type, being
essential for elongation of the peptide chain in
non-ribosomal peptide synthesis. The C domain
catalyzes the peptide bond formation between the
aminoacyl substrates attached to T domains of ad-
jacent modules (Stachelhaus et al. 1998). According
to the thiotemplate model (Stein et al. 1996), C
domains comprise a donor and an acceptor site.
The latter binds the aminoacylated cofactor which
acts as nucleophile while the donor harbors the
cofactor of the upstream module which is loaded
with an aminoacyl or peptidyl group. The C domain
catalyzes the nucleophilic attack which results in the
formation of a peptide bond between the activated
acyl group and the free amino (or imino or hydroxyl)
group of a second amino acid (de Crécy-Lagard et al.
1995; von Döhren et al. 1997; Fig. 15.7). During
assembly of the peptide chain in an N- to C-terminal
direction the intermediates remain bound to the
enzyme (Roskoski et al. 1970).
The C domains appear to be enzymes to be
found exclusively in NRPSs. Solely the highly con-
served core motif HHxxxDG (C3; see Table 15.2)
resembles the active site motif of acyl transferases
(de Crécy-Lagard et al. 1995; Marahiel et al. 1997).
Mutation of the second histidine residue in this
motif abolishes the condensation reaction (Sta-
chelhaus et al. 1998).
As described above, the specificity of an NRPS
module is principally controlled at the adenyla-
313
Fig. 15.7. The condensation do-
O
H main catalyzes peptide bond
N formation between the acyl
S
O R2 attached to the T domains of
adjacent modules
tion/pantetheinylation step. However, by means
of mischarged donor and acceptor aa-S-Ppant T
domains Belshaw et al. (1999) demonstrated that
there is also a selectivity of the C domain at the
acceptor site. Both donor and acceptor site exhibit
strong stereoselectivity while the acceptor site also
shows significant side-chain selectivity (Lautru
and Challis 2004). Rausch et al. (2007) recently
reviewed the current knowledge of subtypes of
the C domain. LDL-catalysts catalyze the conden-
sation of two L-amino acids. DCL-catalysts
are C-domains that are located immediately
downstream of epimerization domains and
are D-specific for the upstream donor and L-
specific for the downstream acceptor, and there-
fore form a peptide bond between a D- and
an L-residue (Clugston et al. 2003). Dual
epimerization/condensation (E/C) domains are
DCL catalysts with epimerase activity, which cata-
lyze the epimerization of an L-residue and subse-
quently promote the condensation of this amino
acid with the amino acid attached to the upstream
module. E/C-domains exhibit a second His-motif
(HH(I/L)xxxGD) which is located near the N-
terminus (Balibar et al. 2005). Heterocyclization
(Cyc) domains catalyze peptide bond formation
and subsequent cyclization of cysteine, serine and
threonine which results in formation of a five-
membered heterocyclic ring (Sieber and Marahiel
2005). Cyc domains are related to C-domains but
the active site motif is replaced by the motif
DxxxxD in which the aspartate residues are criti-
cal for both reactions performed by this domain
(Keating et al. 2002).
4. Modifying Domains
Typically NRPs contain unusual or modified amino
acids. Fungal non-ribosomal peptides often con-
tain modifications such as N-acylation and C-
terminal reduction (peptaibols), N-methylations
(cyclosporin, enniatin) and site-specific epimeriza-
tions. N-methylations and epimerizations are con-
314 Katrin Eisfeld
ducted by particular domains while the latter can
also be conducted by distinct enzymes. Here,
N-methylation (N-MTase) domains and epimeriza-
tion (E) domains are described.
a) N-Methylation Domain
N-Methylated peptides produced by fungi include
pharmacological interesting peptides such as cy-
closporin, members of the enniatin family and
aureobasidin A. The genes encoding cyclosporin
synthetase, enniatin synthetase and peramine syn-
thetase have been studied (see Table 15.1), but
N-methylated amino acids are also found in
PF1022, beauvericin, tentoxin, cyclopeptin and
omphalotins and there are predicted N-methyla-
tion domains in genomes of e.g. Magnaporthe
grisea, Aspergillus flavus, A. nidulans and Phaner-
ochaete chrysosporium (Peeters et al. 1988; Lieber-
mann and Ramm 1991; Sterner et al. 1997;
Weckwerth et al. 2000; Eisfeld, unpublished
data). N-Methylation of peptides contributes to
their biological activity and to peptide bond stabi-
lization against proteolytic cleavage (Marahiel
et al. 1997).
N-Methylation of amino acids during non-
ribosomal peptide synthesis occurs at the thioe-
ster stage prior to peptide bond formation
(Zocher et al. 1983, 1986; Schauwecker et al.
2000; Weckwerth et al. 2000). The N-methyltrans-
ferase activity is determined by N-MTase domains
(420 amino acids) which are inserted between
core motifs A8 and A9 of the corresponding
A domains (Fig. 15.4, Table 15.2). The 40 -phos-
phopantetheine-bound amino acids are substrates
for the corresponding methyltransferase.
S-Adenosyl-methionine (SAM) serves as methyl
donor. The N-MTase domains of NRPSs catalyze
the transfer of the methyl residue onto the cova-
lentely bound aminoacyl (or peptidyl) group,
releasing S-adenosyl-L-homocysteine (AdoHcy)
as a reaction product (Lawen and Zocher 1990;
Dittmann et al. 1994; Zocher and Keller 1997;
Glinski et al. 2002). Recently, Hornbogen et al.
(2007) demonstrated that AdoHyc acts as a regu-
lator of enniatin synthesis by inhibiting the for-
mation of desmethyl enniatin. However, in the
absence of SAM, non-methylated products form
at a reduced rate (Glinski et al. 2001), indicating
that N-methylation is not obligatory for the follow-
ing condensation reaction.
N-MTase domains contain four signature core
motifs including a glycine-rich sequence (motif I;
I II/Y IV V
I VLEIGTGSGMIL
II/Y SYVGLDPS AdoMet binding activity
IV DLVVFNSVVQVFTPPEYL
V ATNGHFLAARA
Fig. 15.8. Position and function of conserved motifs of the
methylation domain
VL(D/E)GxGxG), which is similar to the SAM-
binding site of the heteologous class of cosub-
strate-dependent methyltransferases (Hacker
et al. 2000; Fig. 15.8). Aside from structural fea-
tures typical of other AdoMet-dependent methyl-
transferases, N-MTase domains of NRPS share
only weak sequence similarity to other methyl-
transferases (Billich and Zocher 1987; Burmester
et al. 1995; Hacker et al. 2000).
b) Epimerization Domains and Amino Acid
Racemases
Besides N-methylation, incorporation of D-amino
acids is also a common feature of non-ribosomal
peptide syntheses in fungi. The corresponding
NRPSs mostly contain an epimerization (E) do-
main. These domains are about 420 amino acids
in size. E domains strongly resemble condensa-
tion domains but can be distinguished by the
presence of seven signature sequences character-
istic for these domains (Table 15.2). In a linear
NRPS, E domains are located at the C-terminus of
the respective module’s T domain. Two types
of E domains can be distinguished: E domains
originating from elongation modules (peptidyl-
E domains) and E domains of initiation modules
(aminoacyl-E domains). Epimerization of ami-
noacyl and peptidyl intermediates takes place at
the thioester stage. The reaction is reversible, thus
an equilibrium of both isomers is established
(Stachelhaus and Walsh 2000). The condensation
reaction is involved in the control of stereospecif-
ity by catalyzing solely the elongation of the
D-isomer (Belshaw et al. 1999; Clugston et al.
2003). However, the active site of the E domain
selectively stabilizes the D-isomer in the E domain
of gramicidin S synthetase. Therefore, stereospe-
cifity is not only controlled in the subsequent
reaction but also at the epimerization stage
(Stachelhaus and Walsh 2000).
 Non-Ribosomal Peptide Synthetases of Fungi
Incorporation of a D-amino acid may also be
determined by the specificity of the A domain.
In this case, an external racemase provides the
D-amino acid. Well known examples in fungal
peptide synthesis are alanine residues in cyclo-
sporin and HC toxin. Hoffmann et al. (1994)
characterized the racemase involved in cyclo-
sporin formation. The enzyme catalyzes the re-
versible racemization of alanine and requires
pyridoxal phosphate as the exclusive cofactor.
In Cochliobolus carbonum, the TOXG gene en-
codes the alanine racemase required for synthe-
sis of D-Ala for incorporation into HC toxin
(Cheng and Walton 2000).
5. Termination Domains
The final step in non-ribosomal peptide synthesis
is the release of the covalentely bound peptide
from the enzyme. This step is catalyzed by
specialized C-terminal domains which on the
one hand must accept the mature product and
on the other hand must recognize the full-length
peptide chain to prevent hydrolysis of incomplete
peptides (Schneider and Marahiel 1998). In
bacteria, C-terminal thioesterase (TE) domains
commonly release the final product. However,
termination of non-ribosomal peptide synthesis
may also be catalyzed by reductase (R) domains
or condenation domains. The latter are found
mainly in fungal NRPSs (Keating et al. 2001).
a) Thioesterase Domain
In bacterial NRPSs, the release of the nascent
peptide is mostly catalyzed by a thioesterase
(TE) domain which comprises about 250 amino
acids. This domain has homology to thio-
esterases found in the fatty acid metabolism
(Pazirandeh et al. 1991). After transfer of the
peptidyl intermediate onto a catalytic serine or
cysteine the TE domain leads to the release of
the nascent peptide by hydrolysis, cyclization
or oligomerization (Samel et al. 2006). Most fun-
gal NRPSs, however, lack TE domains (Walton
2004). One exception is the ACV synthetase
which is highly conserved between bacteria and
fungi. There is also a terminal TE domain in an
uncharacterized Phanerochaete chrysosporium
NRPS and in A. nidulans TdiA (see Table 15.1;
Balibar et al. 2007).
315
b) Reductase Domain
A terminal aldehyde or alcohol group has been
found in various non-ribosomally produced
peptides, for example peptaibols. These result
in liberation of the nascent peptide from its
covalent linkage to the enzyme by a reductase
(R) domain. R domains (400 amino acids in
size) share homology with NAD(P)H-dependent
reductases, e.g. in the yeast biosynthetic pathway
for lysine synthesis (Guo et al. 2001; von Döhren
2004).
c) Condensation Domain
In fungi, most NRPSs carry a C-terminal conden-
sation domain which results in cyclization of the
peptide product. Chain termination in this case is
accomplished by amide bond formation. For ex-
ample, in cyclosporin synthesis the terminal C
domain has been assumed to couple the free
amino group of D-Ala1 to the carboxyl group of
L-Ala11. However, there is neither direct evidence
for this reaction nor a lack of evidence for an acyl-
O-C domain intermediate (Keating et al. 2001).
Enniatin synthesis, HC-toxin synthesis and the
synthesis of PF1022A are predicted to follow an-
other mechanism. In analogy to acyl transferase 1,
a direct nucleophilic attack on the thioester bond
was suggested (Scott-Craig et al. 1992; Haese et al.
1993; Pieper et al. 1995; Shaw-Reid et al. 1999;
Weckwerth et al. 2000). The enniatin synthetase
carries a terminal didomain T-C module replacing
the TE domain and allowing the successive accu-
mulation of oligomeres on the T domain prior to
the cyclization mediated by the C-domain (Feifel
et al. 2007).
In LPS1, the ergotamine synthetase of Clavi-
ceps puprurea, a domain with limited similarity to
both, C and cyclization domains of NRPS is locat-
ed at the carboxy terminal side. This domain dif-
fers in the highly conserved motif C3, suggesting a
special mechanism in the final step of the D-lyser-
gyl-peptide lactam synthesis where a diketopiper-
azine is formed from the Phe and Pro moieties
(Walzel et al. 1997).
III. Distinctions Between Fungal
and Bacterial NRPSs
Although fungal and bacterial NRPSs share the
domain architecture typical for these enzymes,
there are some characteristics of fungal peptide
316 Katrin Eisfeld
synthetases that differ from those of bacterial ori-
gin. As described above, conserved sequence
motifs strongly resemble each other in bacteria
and fungi, but also differ to some extend and the
“non-ribosomal code” established with sequence
data from bacterial NRPSs cannot be (fully) ap-
plied to fungal systems (von Döhren 2004;
Schwecke et al. 2006).
The most obvious difference between fungal
and bacterial NRPSs is that most fungal peptide
synthetases consist of one polypeptide chain while
bacterial NRPSs in most cases consist of more
than one protein (Weber et al. 1994). Initially,
this was thought to be characteristic for fungal
NRPSs and still applies to most of them. The
ergot peptide system, however, operates with two
interacting multienzymes, lysergyl peptide syn-
thetase 1 (Lps1) and lysergyl peptide synthetase
2 (Lps2; Riederer et al. 1996). A model of two
interacting NRPSs has also been proposed for
ampullosporin synthesis in Sepedonium ampul-
losporum (Reiber et al. 2003).
Most bacterial NRPSs carry a TE domain at
the C-terminus (Keating et al. 2001). In fungi, TE
domains are rare and mostly a C-terminal C do-
main is found. A TE domain is found in the ACV
synthetase, but this enzyme is an exception as it
shares a very high homology to bacterial ACV
synthetases. It has been suggested that the peni-
cillin biosynthetic gene cluster derived from pro-
karyotic origin and has been transmitted by
horizontal gene transfer (Aharonowitz et al.
1992). Furthermore, typical for fungal NRPSs is
that they often contain D-amino acids, but the
corresponding NRPSs contain no epimerization
domain (Doekel and Marahiel 2001). In these
cases, external epimerases catalyze the formation
of the D-amino acid which is then incorporated by
an adenylation domain with substrate specificity
for the D-isomer (see above). External epimerases
have not yet been described for bacterial NRPSs
where this reaction is typically carried out in cis.
A lot of modifications carried out subsequent to
the assembly of the peptides have been described
for bacterial NRPs. These reactions include halo-
genations, hydroxylations and glycosylations.
The chemical modifications of the peptides are
catalyzed by enzymes which are associated with
the corresponding gene cluster (reviewed by
Samel and Marahiel 2008). Such enzymes as-
sociated with fungal NRPSs have not yet been
described.
Non-ribosomal peptide synthesis has been
studied mainly in bacteria and at lot of infor-
mation about bacterial NRPSs is available. Apart
from that, these enzymes have been described
mostly for filamentous ascomycete fungi. As
described above, many ascomycetes produce
NRPs which are useful in medicine (penicillin,
cyclosporine), or are involved in pathogenesis
(enniatin, HC toxin, AM toxin, gliotoxin),
act as siderophores for iron acquisition or in
iron storage or possess yet unknown activities
(Stachelhaus and Marahiel 1995; Kleinkauf and
von Döhren 1996; Schwarzer and Marahiel 2001;
Haas 2003; von Döhren 2004). The first fungal
NRPS genes examined (e.g. ACV synthetase,
HC toxin synthetase, cyclosporin synthetase and
others) were large genes which contained no
introns (Smith et al. 1990; Gutierrez et al. 1991;
Scott-Craig et al. 1992; Weber et al. 1994). There-
fore, it was suggested, that these genes derived
from horizontal gene transfer from bacteria. The
fact, that fungal NRPS genes are intron-less is
still true for many NRPS genes but meanwhile
also many NRPS genes have been discovered
that bear introns. Some examples are shown in
Table 15.3. The first NRPS gene identified which
is interrupted by at least two introns was a
peptide synthetase gene in Metarhizium aniso-
pliae (Bailey et al. 1996). However, the number
of introns in ascomycetous NRPS genes ranges
from one to seven which still is a very low
intron content, considering the enormous size
of the genes and compared to other genes.
However, in A. fumigatus, ten out of the 14
NRPS sequences identified contain introns with
NRPS1 having the most with six (Cramer et al.
2006).
Table 15.3. Selected NRPS genes of ascomycetes and the
number of introns contained
Producer NRPS Number of
gene introns
Claviceps purpurea CPPS2 2
Neotyphodium lolii lpsA 2
Metarhizium anisopliae PESA 2
Alternaria brassicae AbrePsy1 7
Aspergillus nidulans SIDC 1
A. fumigatus SIDC 1
Schizosaccharomyces SIP1 1
pombe
Magnaporthe grisea SSM1 1
 Non-Ribosomal Peptide Synthetases of Fungi
IV. Non-Ribosomal Peptide Synthesis
in Basidiomycetes
Most fungal NRPSs described so far derive from
ascomycetes. Basidiomycetes are also well known
as producers of interesting natural compounds
and possess a manifold secondary metabolism
(Lorenzen and Anke 1998). Peptides produced
by basidiomycetes include for example the toxic
amanitines and phalloidines (Amanita sp.; Vetter
1998), the immunosuppressive cycloamanides
(Amanita phalloides; Chiang et al. 1982) and the
nematicidal omphalotins (Omphalotus olearius;
Sterner et al. 1997). However, there is not much
information about non-ribosomal peptide synthe-
sis in basidiomycetes.
The bicyclic amatoxins and phallotoxins are octapeptides
and heptapeptides, respectively. These peptides were
thought to be produced non-ribosomally. However, se-
quencing of Amanita bisporogena revealed that this fun-
gus contains no NRPS genes but two genes, AMA1 and
PHA1, which directly encode a-amanitin and phallacidin.
Both peptides are synthesized as proproteins from which
they are predicted to be cleaved by a prolyl oligopeptidase
(Hallen et al. 2007). Hallen et al. suggested that fungi of the
Amanita sp. have a broad capacity to synthesize cyclic
peptides ribosomally as AMA1 and PHA1 are also present
in other toxic species. Amanita virosa produces virotox-
ins, toxic peptides which are solely found in this fungus.
The structure of viroisin, the main virotoxin, is in part the
same as in phallotoxins but contains D-serine instead of L-
cysteine and two unnatural amino acids (2,3-trans-3,4-
dihydroxy-L-proline and 2’-(methylsulfonyl)-L-trypto-
phan). It remains to be elucidated whether this toxin also
is synthesized ribosomally.
However, NRPS genes have been found in other
basidiomycetes. Siderophore biosynthesis has
been investigated in the ustilaginomycete Ustilago
maydis and in the homobasidiomycete Omphalo-
tus olearius. U. maydis produces ferrichrome and
ferrichrome A (Budde and Leong 1989). The
corresponding siderophore synthetases have
been identified. SID2 and FER3 encode the ferri-
chrome synthetase and the ferrichrome A synthe-
tase, respectively (Yuan et al. 2001; Eichhorn et al.
2006). A putative ferrichrome A synthetase gene
has also been found in O. olearius. The NRPS gene
encoding the ferrichrome A synthetase in this
fungus (FSO1) was the first NRPS gene described
in a higher basidiomycete. The basidiomycete fer-
richrome-type siderophore synthetase possesses
317
high levels of similarity to siderophore synthe-
tases of ascomycetes.
Interestingly, FSO1 is interrupted by a total of 48
introns (Welzel et al. 2005). Although a high number
of introns is very unusual for NRPS genes, basidio-
mycete genes generally are interrupted by many
introns (Larrondo et al. 2004; Martinez et al. 2004).
Two more NRPS genes have been identified in O.
olearius: PSO3 and PSO4 (Welzel 2005; Eisfeld, un-
published data). Both NRPSs have unusual domain
architectures and contain a relatively high number
of introns. PSO4 consists of one minimal module
and an additional C domain at its N-terminus
(CATC). PSO3 is displayed in Fig. 15.2. The 15 656-
bp gene is disrupted by 25 introns. Noticeably,
introns often interrupt conserved sequence motifs.
Attempts to identify the products of these NRPSs
have been unsuccessful so far.
The recent annotation of several basidiomycete genomes
facilitates the search for NRPS genes in these fungi. In U.
maydis, besides the siderophore synthetases a partial open
reading frame that contains the N-terminal fragment of an
NRPS was identified (um10543) which has the predicted
domain architecture ATC-ATC-ATC and contains at least
12 introns (Bölker et al. 2008). The Coprinus cinerea ge-
nome seems to comprise one NRPS gene, CC1G_04210,
with a length of 7782 bp, which is interrupted by 14 introns
(Blast searches at the Broad Institute). The truncated mod-
ules (TC) suggest that the deduced peptide synthetase
belongs to the iterative NRPSs (Fig. 15.9). CC1G_04210 is
clustered with an L-ornithine monooxygenase gene, there-
fore this NRPS is likely to encode a siderophore synthetase. L-
Ornithine monooxygenases comprise the first step in syn-
thesis of siderophores of the hydroxamate type. The domain
architecture resembles more that of NRPSs of the coprogen
type than that of the ferrichrome type which in ascomycetes
is mostly ATC-TC or ATC-TTC (Oide et al. 2006). In O.
olearius and in several ascomycetes the siderophore synthe-
tase genes are also clustered with the L-ornithine monoox-
ygenase-encoding gene (Haas 2003; Welzel et al. 2005).
Phanerochaete chrysosoprium was predicted to con-
tain seven NRPS genes (Martinez et al. 2004). However,
Blast searches at NCBI or JGI could only identify one
NRPS. This gene encodes a dimodular NRPS with the
first module comprising an N-MTase domain. Interesting-
ly, a terminal Te domain is found in this NRPS (Fig. 15.9),
which is rare in fungal NRPSs. In silico analyses lead to the
assumption that the P. chrysosporium NRPS gene is inter-
rupted by at least five introns (Eisfeld, unpublished data).
In the genome of the stem rust fungus, Puccinia graminis,
one monomodular NRPS (ATC) can be identified
performing Blast searches at NCBI and this gene is inter-
rupted by three predicted introns. In the Laccaria bicolor
genome no NRPS-encoding gene could be identified so far.
Comparison of conserved sequence motifs in
basidiomycete NRPSs reveals, that sequences show
318 Katrin Eisfeld

O. olearius Fso1
A TC A TC A TCTCTC

Pso3
C A T A TC A TE C A TE

Pso4
C A TC

C. cinerea CC1G_04210
A TCTCTC

P. chrysosporium n.a. A M T C A TTe

U.maydis Sid2 A TC A TC A TCTC

Fer3 A TC A TC A TCTCTC

UMO5245.1 A TC A TC A TC

P. graminis n.a. A TC

Fig. 15.9. NRPSs in basidiomycetes

Table 15.4. Selected NRPS genes of basidiomycetes and long, occurred at an early stage in evolution at
the number of introns contained least 500106 years ago (Taylor and Berbee
Producer NRPS gene Number of 2006). According to the “intron early theory” a
introns near intron-less state of diverse eukaryotes seems
to be due to the massive loss of ancestral introns,
Puccinia graminis Not annotated 2 (in silico
analysis) also bolstering the idea of complete intron loss in
Ustilago maydis SID2 0 prokaryotes (reviewed by Roy and Gilbert 2006).
FER3 1 This might be also reflected in the intron content
UMO5245.1 8 of fungal NRPS genes.
Coprinus cinerea CC1G_04210 14 (predicted)
Phanerochaete Not annotated
chrysosporium
Omphalotus olearius FSO1 48
PSO3 25 V. Physiological Significance of Peptides
PSO4 3
The activities of peptide products with respect
to interactions with other organisms have been
much more variations than those of bacterial origin, extensively studied. For example, the mode of
even within one species (Eisfeld, unpublished data). action of cyclosporin and penicillin and their deri-
While amplification of NRPS gene fragments using vatives is very well documented. However, inter-
degenerate primers deduced from core motifs often organismal activities mostly are not the primary
is successful in bacteria and in ascomycetes, se- function of NRPs and the physiological signifi-
quence variations may hamper or even impede the cance of most of the peptides is unknown
amplification of yet unknown NRPSs in basidiomy- (Turgeon et al. 2008). Among the few well studied
cetes using this approach. functions of small peptides is the function as
It is particularly noticeable that the homoba- siderophores in complexing Fe3+. Siderophores
sidiomycete NRPS genes are interrupted by a of the ferrichrome and of the coprogen type have
higher number of introns than those of U. maydis been described in many fungi as for example
and P. graminis. The P. graminis NRPS-encoding A. fumigatus, A. nidulans, U. maydis, O. olearius,
gene seems to be interrupted by two introns S. pombe, and various phytopathogenic fungi
(Table 15.4). The divergence of ustilaginomycetes (M. grisea, C. heterostrophus, F. graminearum,
(including U. maydis and P. graminis) from hyme- Alternaria brassicicola, etc.) and an additional role
nomycetes, to which the homobasidiomycetes be- as pathogenicity factor has been demonstrated for
 Non-Ribosomal Peptide Synthetases of Fungi
some of them (Eisendle et al. 2003, 2006; Hof et al.
2005; Welzel et al. 2005; Oide et al. 2006, 2007;
Schwecke et al. 2006). The functions of sidero-
phores were reviewed very recently by Johnson
(2008) and Haas et al. (2008), and Schwecke
et al. (2006) compared peptide synthetases of the
ferrichrome type. In brief, siderophores play very
diverse roles. Their primary function is the acqui-
sition of iron under iron limited conditions and
the storage of iron. Some siderophores are also
required for resistance to oxidative stress, asexu-
al/sexual development or virulence.
There are more non-ribosomally synthesized
peptides which act as virulence factors during
plant pathogenesis. These peptides include AM
toxin from Alternaria alternata (Panaccione et al.
1992; Johnson et al. 2000) which causes the apple
pathotype, and the maize pathogenic HC toxin
from Cochliobolus carbonum (Scott-Craig et al.
1992; Jones and Dunkle 1995), and also enniatin
from Fusarium spp. (Herrmann et al. 1996). A
possible function for petaibols as virulence factor
has also been described. Peptaibol-producing fungi
of the genus Sepedonium infect fruiting bodies of
basidiomycetes of the order Boletales. The peptai-
bols are thought to facilitate the invasion process
by damaging host tissues in the basidiomycetes
and also in insect invasion processes (Matha et al.
1992; Engelberth et al. 2001).
Endophyte toxins may play a protective role
for their hosts. Peramine for example is a potent
insect feeding deterrent. This pyrrolopyrazine
is the product of a two-module NRPS and is
synthesized by Epichloë/Neophytodium mutual-
istic endophytes (Tanaka et al. 2005). Some
perennial ryegrass/Neotyphodium sp. symbiota
accumulate ergovaline which reduces the appeal
to rabbits. Rabbits prefer plants that are endo-
phyte-infected but free of ergot alkaloids over
endophyte-free plants. Therefore, the accumu-
lation of ergot alkaloids seems to counteract
the added appeal of endophyte-infected plants
(Panaccione et al. 2006).
In order to characterize the function of all non-
ribosomally produced peptides in one fungus, Tur-
geon et al. (2008) deleted each of the 12 NRPS genes
in C. heterosprophus. Four of the genes are con-
served in ascomycete genomes, and these genes
were also the only ones yielding phenotypes. Two
of the genes (NPS6, NPS2) are siderophore synthe-
tases. Deletion of NPS10 led to an alteration in the
morphology of the colonies and hypersensitivity
319
to various stresses. Deletion of NPS4 led to loss
of hydrophobicity, a phenotype also described
for the homologous genes in A. brassicicola
(AbNPS2) and G. zeae (Kim et al. 2007; Turgeon
et al. 2008). Turgeon et al. (2008) speculated that
the corresponding NRPS produces a metabolite
similar to acuminatum, a cyclodepsipeptide which
has surfactant properties or a peptide that regulates
the production of hydrophobins (Tobiasen et al.
2007). In A. brassicicola, deletion of the homolo-
gous gene led not only to a decreased hydrophobi-
city phenotype but also to an abnormal spore cell
wall morphology and decreased spore germination
rates, indicating that AbNPS2 plays an important
role in development and virulence (Kim et al.
2007). Consequently, these studies showed that
NRPs may play a role in basic biological processes
such as nutrient acquisition and sexual and asexual
development. However, the physiological function
of many of them still remains to be elucidated.
VI. Examples for NRPS Gene Clusters
and Cluster Evolution
In contrast to genes involved in primary metab-
olism, in fungi secondary metabolite genes are
often organized in gene clusters (Keller and
Hohn 1996). These genes are located adjacent to
one another and are co-regulated. This is also the
case for those pathways involved in non-ribo-
somal peptide synthesis (Walton 2000; Gardiner
et al. 2004; Haarmann et al. 2005; Cramer et al.
2006; Zaleta-Rivera et al. 2006; Johnson et al.
2007). Models to explain this phenomenon in-
clude the suggestion that these genes derived
from horizontal gene transfer from prokaryotes.
More recent evidence suggests regulatory mech-
anisms as evolutionary driving force. Also, the
clustering increases the probability of co-mobili-
zation. Thus, there is a selective advantage to the
gene cluster itself (Walton 2000; Hoffmeister and
Keller 2006). There are some examples of hori-
zontal transfer of fungal nuclear genes, and hori-
zontal gene transfer has often been suggested as
an explanation for the discontinuity in distribu-
tion of some fungal secondary metabolite genes
when they are present only in some isolates of a
species and are absent from others (Walton
2000). The horizontal transfer of an entire chro-
mosome between two vegetative incompatible
320 Katrin Eisfeld

isolates of the same species has been demon- comycete lineages (Patron et al. 2007). In M. grisea, the
strated by He et al. (1998). This may explain the avirulence gene ACE1 which codes for a hybrid PKS-NRPS
is organized in a cluster of 15 genes. Related clusters were
origin of supernumerary chromosomes in fungi found in several other ascomycetes, e.g. Chaetomium glo-
which has been observed in several species. For bosum and A. clavatus, which appear to have obtained
example, Wang et al. (2003) demonstrated that a the cluster by horizontal gene transfer form a donor close-
dispensable (CD) chromosome exists in the in- ly related to M. grisea (Khaldi et al. 2008).
sect pathogenic fungus, M. anisopliae. The genes
conferring the ability to produce destruxin are
most likely located on this chromosome (Wang
et al. 2003). The cyclic peptide synthetase gene A. Penicillin and Cephalosporin Biosynthesis
encoding the AM toxin synthetase in Alternaria
Penicillins and cephalosporins are b-lactam con-
alternata also resides on a CD chromosome. Loss
taining antibiotics produced both of prokaryotes
of this chromosome leads to loss of AM toxin
and eukaryotes. Penicillin G production is de-
production and pathogenicity (Johnson et al.
scribed solely for fungi (e.g. P. chrysogenum,
2001). The O. olearius peptide synthetase gene
P. nalgiovenese, P. griseofulvum, P. dipodomys,
PSO3 also is located on a CD chromosome
P. flavigenum, A. nidulans). Cephalosporin is
(Eisfeld, unpublished data).
produced by both bacteria and fungi, for exam-
Some NRPS gene clusters are widely distributed in differ- ple the Ascomycetes Acremonium chrysogenum
ent ascomycete lineages. For example, putative epipo- and Kallichroma thetys and the Actinomycetes
lythiodioxopiperazine (ETPs) gene clusters are present in Streptomyces clavuligerus, Streptomyces lipmanii
14 ascomycete taxa. The gene content of the homologous and Nocardia lactamdurans. Biosynthesis and
clusters is not identical but common genes were identified
in these 14 species. The ETP gene clusters appear to have a gene clusters for b-lactam antibiotics and the
single origin and have been inherited relatively intact control of their expression were recently
rather than assembling independently in the different as- reviewed by Brakhage et al. (2005) and Liras

a-aminoadipic acid + cysteine + caline

ACV synthetase
pcbAB
H2N H SH
N
H
O
COOH CH3
N
O H COOH
H

IPN cyclase
pcbC

H2N H S
N CH3
CH3
COOH
O Isopenicillin N
N
O COOH

Acyl- PenN epimerase cefD

transferase DAOC synthase, DAC hydroxylase cefE, cefEF
penDE Carbamoyl-transferase cmcH
Acetyl-transferase cefG
H S H 2N H S
N CH3 N
O CH3 O O
COOH
N N CH2OCCH3
O COOH O
COOH

Penicillin G Cephalosporin C

Fig. 15.10. Biosynthetic pathways of b-lactam antibiotics (modified from Liras and Martin 2006)
 Non-Ribosomal Peptide Synthetases of Fungi
Penicillium chrysogenum
Penicillium verrucosum
Aspergillus nidulans
cefT
Acremonium chrysogenum
pcbC
Strepromyces clavuligerus
pcbC
Lysobacter lactamgenus
Fig. 15.11. Excerpt of penicillin/cephalosporin gene cluster of several fungi and bacteria. The NRPS gene is indicated as a
black arrow. The arrows indicate the orientation of transcription (modified from Liras and Martin 2006)
and Martı́n (2006). In brief, the first two enzymat-
ic steps are common to all b-lactam producers.
First, a NRPS (ACV synthetase, encoded by
pcbAB) catalyzes the formation of the tripeptide
d-(L-a-aminoadipyl)-L-cysteinyl-D-valine which in
the second step is converted to the bicyclic iso-
penicillin N by the action of isopenicillin N (IPN)
synthase (also named ACV cyclase), encoded by
the pcbC gene (Fig. 15.10). This is the branch
point for various penicillin and cephalosporin
pathways. Fungal penicillin producers contain a
third gene, penDE, which encodes an isopenicillin
N acyltransferase that leads to the formation of
penicillin G by hydrolyzation of the a-aminoadi-
pic sidechain of isopenicillin N and introduction
of phenylacetyl-CoA. For cephalosporin C synthe-
sis, a set of genes is needed which encode the
enzymes responsible for isomerization of isopeni-
cillin N (cefD), conversion of the thiazolidine ring
of penicillin N to a dihydrothiazine ring and hy-
droxylation at C-3 (cefEF in fungi, cefE and cefF in
bacteria) and acetylation (cefG).
The genes responsible for b-lactam biosynthe-
sis are clustered in all producers (pro- and eukar-
yotes). Figure 15.11 shows the structure and
orientation of some of the genes. The pcbC and
pcbAB genes are always located next to each other.
In penicillin-producing fungi, the penDE gene is
directly adjacent. The epimerization reaction to
form the D-isomer penicillin N in Acremonium is
encoded by two genes, cefD1 and cefD2 (while in
bacteria only one gene, cefD, is necessary for this
reaction). cefEF and cefG, whose gene products
are involved in the final steps of cephalosporin
synthesis, are linked and located on a different
chromosome.
321
pcbAB pcbC penDE
pcbAB
pcbAB pcbC penDE
pcbAB pcbC cefD2 cefD1
pcbAB
pcbAB
It was suggested that the b-lactam gene cluster
has bacterial origin and was transmitted by hori-
zontal gene transfer (Weigel et al. 1988; Aharono-
witz et al. 1992). This theory is supported by the
fact that the structure and orientation of the genes
in the penicillin gene cluster is the same in both
Aspergillus (MacCabe et al. 1990) and Penicillium
(Laich et al. 1999). Furthermore, high homologies
exist between the bacterial and fungal genes that
are shared in both groups (ACV synthetase, IPN
synthase, CefE, CefF) and which exceed those of
genes belonging to primary metabolism. In addi-
tion, introns are lacking in pcbAB, pcbC and
cefEF, while genes absent in bacterial clusters do
contain introns (Liras and Martı́n 2006). There-
fore, horizontal gene transfer of pcbAB and pcbC
either took place about 370106 years ago, before
the split between Aspergillus and Penicillium
occurred or multiple gene transfer events took
place (Weigel et al. 1988; Aharonowitz et al.
1992). Laich et al. (2002) found a cluster of genes
almost identical to that in P. chrysogenum in
several other Penicillium spp. (P. nalgiovense,
P. griseofulvum) while a truncated cluster
(consisting of pcbAB and lacking pcbC and
penDE) is present in P. verrucosum. Therefore,
the penicillin gene cluster or just parts of the
cluster as in P. verrucosum may have been lost
throughout the evolution from a common ances-
tor (Fig. 15.11).
B. Ergot Alkaloid Biosynthetic Gene Clusters
The ergot alkaloids and their synthesis have been
extensively studied and were recently reviewed, for
322 Katrin Eisfeld
DMAPP + tryptophane
dmaW
DMAT
clavine alkaloids
cloA
D-Lysergic acid
lpsB + lpsA1 lpsB + lpsA2
ergotamine ergocristine
Fig. 15.12. Biosynthetic pathway of ergot alkaloids in
Claviceps purpurea (modified from Haarmann et al. 2005)
example by Lorenz et al. (2007) and Schardl et al.
(2006). Ergot alkaloids share a four-membered
ergoline ring but differ in the number, type and
position of the side-chains. These pharmacological
interesting mycotoxins are among others pro-
duced by fungi of the relatively distant taxons
Clavicipitaceae and Epichloë endophytes of
grasses and A. fumigatus.
As ergoline alkaloids which are synthesized by
epibiotic fungi present on different plant species
is the topic of Chap. 9, this chapter focuses on the
peptide synthetases involved in ergot alkaloid
synthesis and the evolution of the gene cluster.
The first ergot alkaloid gene cluster analyzed was
that in Claviceps purpurea. The first enzyme
identified was dimethylallyl-tryptophan synthase
(DMATS) which catalyzes the first step in the
pathway (prenylation of tryptophan with forma-
tion of dimethylallyltryprophan; DMAT; see
Figs. 15.12, 15.13; Gebler et al. 1992). The gene
encoding this protein, dmaW, was identified in
C. purpurea (Tsai et al. 1995; Tudzynski et al.
1999) and also characterized in A. fumigatus
(Coyle and Panaccione 2005), Claviceps fusiformis
(Tsai et al. 1995), and Neotyphodium sp. strain
Lp1 (Epichloë typhina x N. lolii; Wang et al.
2004). Using this gene as a starting point, Tud-
zynski et al. (1999) cloned additional genes,
providing evidence for an ergot alkaloid gene
cluster which now comprises 14 genes in a geno-
mic region of 68.5 kb (Correia et al. 2003; Haar-
mann et al. 2005; Lorenz et al. 2007). Besides
dmaW, the function of cloA (conversion of ely-
moclavine to paspalic acid) and others has been
functionally analyzed by heterologous expression
or gene replacement approaches (Tsai et al. 1995;
Correia et al. 2003; Haarmann et al. 2006). The
pathway leading to two different ergot alkaloids is
depicted (simplified) in Fig. 15.12.
The ergot alkaloid gene cluster in C. purpurea
comprises four NRPS genes. The lysergyl peptide
synthetase is a complex consisting of two NRPSs,
LpsB and one of two different versions of LpsA
which are encoded by lpsA1 and lpsA2, respectively.
LpsA from an ergocristine-producing isolate of C.
purpurea differs from that of a previously char-
acterized ergotamine-producing isolate con-
cerning the signature sequences in the first
module leading to the recognition of different
amino acid substrates by the adenylation
domains. The function of the fourth NRPS in
the ergot alkaloid gene cluster of C. purpurea,
lpsC, is still unknown (Haarmann et al. 2005).
The LpsA-encoding gene of Neotyphodium
lolii was also analyzed and compared to that of
C. purpurea. Both fungi produce different ergo-
peptines as most abundant ergot alkaloids. In
N. lolii ergovaline is produced. Damrongkool
et al. (2005) found that a copy of the NRPS
gene was present which at the 5’ end encodes a
partial condensation domain in N. lolii. This
truncated C domain is missing in C. purpurea
LpsA. Both NRPSs have similar signature se-
quences in modules one and three but diverg-
ent signature sequences in module two. This
finding is consistent with the amino acid found
at the corresponding position in the major
alkaloids of these fungi. The LpsA-encoding
genes in N. lolii and C. purpurea both are inter-
rupted by two introns. The position of the
introns is conserved and, furthermore, located
at the same point preceding the first and third
module of the NRPSs. The latter finding sup-
ports the hypothesis that multimodular NRPS
genes arose by duplication of an ancestral mono-
modular NRPS-encoding gene (Damrongkool
et al. 2005).
Claviceps fusiformis and A. fumigatus are rel-
atively distant taxons but both produce clavines.
 Non-Ribosomal Peptide Synthetases of Fungi 323

lpsC easA lpsB cloA easC easD easE easF easG dmaW easH lpsA1 lpaA2

Cp

ap lpsB easA cloA easC easD easE easF easG dmaW

Cf

easH easA easG easF easE lpsB

Nl

easH easA easG easK easL easD easM easN easC dmaW easE easF

Af

Fig. 15.13. Schematic comparison of the ergot alkaloid gene clusters of C. purpurea (Cp), C. fusiformis (Cf ), Neotypho-
dium lolii (Nl ) and A. fumigatus (Af ). NRPS genes in the species are indicated by black arrows. The arrows indicate the
orientation of transcription (modified from Fleetwood et al. 2007; Lorenz et al. 2007)

Therefore, the NRPSs and cloA are dispensable in VII. Conclusions

these organisms. Coyle and Panaccione (2005)
investigated whether the ergot alkaloids of
A. fumigatus have a common biosynthetic origin
with those of the clavicipitaceous family. dmaW
was identified and could be complemented with
the corresponding C. purpurea gene after it had
been deleted. Besides this, five further genes which
are proposed to encode steps in the ergot alkaloid
pathway were detected, but orientation and posi-
tions of several genes differed. This is also the case
for C. fusiformis. In this fungus, nine homologues
of genes identified in the C. purpurea gene cluster
were identified. Interestingly, C. fusiformis con-
tains homologues of cloA and lpsB, but both are
pseudogenes which contain frameshifts and stop
codons. These findings support the proposal that
the ergot alkaloid gene cluster evolved from a
more complex cluster and that during evolution
some genes were lost or inactivated due to muta-
tions and rearrangements. In an ergovaline-
producing Neotyphodium lolii strain, the ergot
alkaloid gene cluster was analyzed and homolo-
gues to all genes found in the C. purpurea
cluster were identified except for two genes
(Fleetwood et al. 2007). Despite conservation
of gene sequences, gene order is substantially
different between the N. lolii, A. fumigatus and
C. purpurea gene clusters (Fig. 15.13). In N. lolii,
several long terminal repeat retrotransposons and
non-autonomous transposable elements were
identified in intergenic regions of the cluster
which could be the reason for rearrangement
events in the overall structure of the cluster
(Fleetwood et al. 2007).
Identification of new NRPS products with poten-
tially interesting biological effects has been
facilitated by genome sequencing projects. Knowl-
edge of regulators of secondary metabolism help
to identify transcriptionally active clusters and
also to specifically manipulate their expression.
Furthermore, silent or cryptic gene clusters may
be activated leading to new compounds. Genetic
engineering using combinatorial biosynthesis
may also lead to novel NRPs.
A. Approaches to Identify New NRPSs
Identification of pharmaceutically interesting nat-
ural products has been merely achieved by screen-
ing technologies. This strategy has been even
more successful with the advent of high-through-
put screening (Wilkinson and Micklefield 2007).
However, evidence is emerging that the genomes
of fungi are richer in genes that are likely to be
involved in the synthesis of secondary metabolites
and many of these metabolites have not been
discovered yet. The capacity to produce NRPs in
some fungi is very high, as information about
fungal genomes revealed. The conservation of
DNA and protein sequences greatly facilitates
identification of NRPS genes in the completed
genomes. For example, Aspergilli genomes com-
prise 14 or more NRPS genes (Cramer et al. 2006),
C. heterosptrophus contains 11 NRPS genes (Lee
et al. 2005) and Fusarium graminearum 15 NRPS
genes (Varga et al. 2005; Tobiasen 2007). The
324 Katrin Eisfeld
genes were identified by in silico analyses and the
ongoing growth of DNA sequence data will lead to
the discovery of more natural-product biosynthe-
sis pathway genes.
The number of putative NRPSs is much greater
than the known peptides described for these
species. One reason for this might be that these
compounds are not produced under laboratory
conditions (Bok et al. 2006; Brakhage et al. 2008).
These “silent” or cryptic biosynthetic genes may
encode the information for potentially useful
metabolites that still await discovery. Silent gene
clusters can be activated for example by heterolo-
gous expression under the control of a defined
promoter or promoters of biosynthesis genes
within the genome can be exchanged by defined
promoters (Brakhage et al. 2008). Bergmann et al.
(2006) identified a silent gene cluster in A. nidu-
lans which contained a gene encoding a hybrid
PKS-NRPS and an activator gene. This activator
gene was retransformed into A. nidulans and
expressed under the control of an inducible pro-
moter. Under inducing conditions, the gene
encoding the PKS-NRPS hybrid synthetase was
transcribed and the metabolite could be identified.
This most promising approach takes into account
that all pathway genes are co-regulated.
Another interesting approach was enabled by
the identification of a global regulator of second-
ary metabolism in Aspergillus spp., LaeA, by Bok
and Keller (2004). Deletion of the gene blocks the
expression of metabolic gene clusters including
that responsible for penicillin production in
A. nidulans while overexpression of laeA triggers
an increased gene expression. Bok et al. (2006)
performed microarrays and demonstrated that
transcribed secondary metabolite gene clusters
can be revealed by profiling laeA deletion and
overexpression mutants. Using this method, the
authors were able not only to identify transcrip-
tionally active clusters but also to specifically
manipulate their expression and characterize a
novel natural product, terrequinone, and its gene
cluster, in A. nidulans.
B. Combinatorial Biosynthesis of New NRPs
A current challenge in NRPS research is to re-
engineer natural products in order to increase or
alter their biological activities. This can be
achieved by the construction and combination of
domains and modules at the genetic level, for
example by exchanging A-T units, artificial fusion
of modules, module swaps and deletions. These
applications have been successful in bacterial sys-
tems especially by work with Streptomyces spp.,
reviewed by Sieber and Marahiel (2005). A recent
chemoenzymatic approach combines synthetic
peptide synthesis with TE domain cyclization
(reviewed by Kopp and Marahiel 2007). However,
combinatorial biosynthesis has not yet been ap-
plied to fungal systems to a large extend. The
establishment of fungal systems for domain ex-
change requires a more profound knowledge of
the domain interactions, A domain specificity and
linker regions between the domains, as identified
in bacterial NRPSs (Mootz et al. 2000; Keating
et al. 2002; von Döhren 2004).
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16 Biosynthesis of Fungal Polyketides

JULIA SCHUEMANN1, CHRISTIAN HERTWECK1

CONTENTS body of knowledge exists on bacterial polyketide

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 biosynthesis, the corresponding fungal systems
II. Ecological Importance and Pharmaceutical are far less explored. Not until 1990 did scientists
Use of Fungal Polyketides . . . . . . . . . . . . . . . . . . . . 331 clone and sequence the first fungal polyketide
III. Biosynthesis of Polyketides . . . . . . . . . . . . . . . . . . 333 synthase (PKS) gene, encoding 6-methylsalicylic
IV. Fungal Polyketide Synthase Classes . . . . . . . . . 334
A. Non-Reducing PKS . . . . . . . . . . . . . . . . . . . . . . . 335 acid synthase (6-MSAS; Beck et al. 1990). While
B. Partially Reducing PKS . . . . . . . . . . . . . . . . . . . 337 the product (6-MSA) has a relatively simple struc-
C. Highly Reducing PKS . . . . . . . . . . . . . . . . . . . . . 337 ture, some highly complex polyketides are also
D. Polyketide Synthase–Non-Ribosomal produced by fungi, for example T-toxin (Yang
Peptide Synthetase Hybrids . . . . . . . . . . . . . . 339 et al. 1996) and the cytochalasins (Binder and
V. Post-PKS Modifications . . . . . . . . . . . . . . . . . . . . . . . 341
VI. Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 Tamm 1973). The derivatisation or total synthesis
VII. Functional Analysis and Engineering of such compounds can be cumbersome or ineffi-
of Fungal Polyketide Biosynthesis . . . . . . . . . . . 344 cient, making pathway engineering approaches
A. Inactivation of PKS Genes . . . . . . . . . . . . . . . . 345 attractive. In the era of whole-genome sequencing
1. Knockout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 we have an insight into the biosynthetic potential
2. Knockdown . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
B. Heterologous Expression of PKS Genes . . 346 of fungi. Even so, yet very few of those identified
C. PKS Pathway Regulation . . . . . . . . . . . . . . . . . 347 PKS genes have been linked to natural products.
VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 Thus, the improvement of fungal molecular and
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 genetic techniques is leading to enormous prog-
ress in understanding and manipulating fungal
polyketide biosynthesis. This review focuses on
I. Introduction the ecological and medical aspects of polyketide
biosynthesis in fungi, the mechanisms involved
in polyketide assembly and approaches to mani-
Fungal secondary metabolism is a rich source
pulate the pathways.
of biologically active compounds. Besides non-
ribosomal peptides and terpenoids, fungi are
known as producers of structurally diverse poly-
ketides. Many of these metabolites are infamous II. Ecological Importance
toxins, such as aflatoxin (Minto and Townsend
and Pharmaceutical Use
1997) and fumonisin (Chu 1991; Sweeney and
Dobson 1999), pigments or virulence factors like of Fungal Polyketides
melanin (Perpetua et al. 1996; Fig. 16.1). However,
fungal polyketides may also be pharmaceutically Polyketides are a class of secondary metabolites
relevant, like the cholesterol-lowering agent lova- that exhibit a broad spectrum of relevant activi-
statin (Hendrickson et al. 1999). While a large ties. Fungal polyketides may serve as precursors
to toxins that have adverse effects on animals or
against plants. Important examples are lovastatin
from Aspergillus terreus (Hendrickson et al. 1999),
1
Leibniz Institute for Natural Product Research and Infection
citrinin from Monascus purpureus (Shimizu et al.
Biology, HKI, Beutenbergstrasse 11a, 07745 Jena, Germany; 2005), fumonisin from Gibberella fujikuroi
e-mail: Christian.Hertweck@hki-jena.de (Proctor et al. 1999), T-toxin from Cochliobolus

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds)
© Springer‐Verlag Berlin Heidelberg 2009
332 Julia Schuemann and Christian Hertweck

O OH OH O
O OH O OH
OH O
HO HO O
HO
O
OH O

Orsellinic acid 6-MSA Citrinin Zearalenone

O O
OH OH
HO O
OH
H3CO O OCH3
HO OH
OH OH O O

THN YWA1
Bikaverin

O COOH
O COOH
HOOC
HO
HOOC O O OH OH
O
OH
HOOC O O OH NH2
Fumonisin
O
O
Squalestatin HOOC COOH
O HO O

O O O
O
OH O OH O
O
O
H
O
HO OH O
MeO
O R
Norsolorinic acid Aflatoxin B1 R = CH3: Lovastatin
(R = H: Compactin)

OH OH O O OH O O OH O O OH O O

T-Toxin

Fig. 16.1. Structures of selected fungal polyketide metabolites

heterostrophus (Yang et al. 1996) and aflatoxin
produced by Aspergillus parasiticus (Feng and
Leonard 1995). These compounds, among others,
are infamous for causing severe damage in food
industry, agriculture and human health. Aflatox-
ins are potent animal and human pathogens
(Eaton and Groopman 1994). They show immuno-
suppressive, mutagenic, teratogenic and hepato-
carcinogenic properties. Aflatoxins are produced
by several species of Aspergillus, contaminating
commodities, corn, peanuts, cotton seed and
tree nuts. The production of aflatoxins probably
protects the producer strain from feeding
competitors.
A similar function in habitat defence but
also in plant destruction might be due to fumoni-
sins, produced by a number of cereal-pathogenic
G. fujikuroi. Fumonisins cause several mycotoxi-
coses, like leucoencephalomalacia in horses, pul-
monary oedema in pigs and liver cancer in rats
(Nelson et al. 1993). Their chemical structure
resembles sphingosine. Fumonisins are competi-
tive inhibitors of sphingosine N-acetyltransferase.
They block complex sphingolipid biosynthesis
and lead to the accumulation of sphingosine
(Moss 1998). G. fujikuroi MP-A is one of the
most common pathogens of maize. It is associated
with diseases of the roots, stalk and ears but addi-
tionally the fungus is frequently present in appar-
ently healthy maize tissues (Munkvold and
Desjardins 1997).
Cytochalasins comprise a miscellaneous
group of fungal polyketides with an impressing
variety of biological activities. Beside cytotoxic
 Biosynthesis of Fungal Polyketides
activities various members have antibiotic, anti-
inflammatory, antitumoral and antiviral activities.
They can affect glucose transport, release of thy-
roid and growth hormones and inhibit cholesterol
synthesis. Because of their defined binding of
actin filaments cytochalasins are used as tools in
cell and molecular biology (Binder and Tamm
1973; Steglich et al. 1997; Lodish et al. 2001).
According to Deacon and others, cytochalasin
production by insect pathogenic fungi, such as
Metarhizium anisopliae, can lead to a lethal toxifi-
cation of insects (Deacon 2001). Cytochalasins
have also been implicated as virulence factors
produced by the plant-pathogenic fungus Phoma
exigua (Scott et al. 1975). Saprophytic fungi like
Penicillium expansum or Xylaria spp. might pro-
duce cytochalasins in order to defend their habitat
and secure their nutrial resources, e.g. wood, leafs,
fruits or seeds.
Polyketides may also function as pigments
like melanin (Perpetua et al. 1996) and bikaverin
(Linnemannstons et al. 2002), or as pathogenicity
factors, e.g. reported for melanin (Perpetua et al.
1996). The pentaketide 1,3,6,8-tetrahydroxy-
naphthalene (THN) plays a key role in the mela-
nisation of various fungi (Butler and Day 1998).
THN is reduced to further intermediates such as
1,8-diydroxynaphthalene (DHN), which under-
goes polymerisation to DHN-melanin (Fig. 16.2).
For plant pathogens, e.g. Colletotrichum lagenar-
ium or Magnaporthe grisea, melanin may support
apressorium formation and penetration of plant
cells (Howard et al. 1991). DHN-melanin likely
plays a protective role in human pathogenic
fungi like Wangiella dermatitidis, Sporothrix
schenckii or Aspergillus fumigatus (Langfelder
et al. 2003). There is strong evidence that melanin
protects the fungus from the host immune system,
e.g. through scavenging reactive oxygen species,
and that it stabilises fungal hyphae for penetration
of host tissue.
T-toxins are a family of C35 to C49 polyke-
tides produced by C. heterostrophus (Kono et al.
OH OH
1 Acetyl-CoA
4 Malonyl-CoA
HO
1,3,6,8-THN
Fig. 16.2. Schematic mechanism of fungal melanin biosynthesis according to Butler et al. (1998)
333
1980, 1981; Kono et al. 1983). They are involved in
virulence on maize (Yoder 1980; Yoder et al. 1997;
Turgeon and Lu 2000).
Various fungal polyketides are medicinally
important or have served as lead structure for
the development of therapeutics. Lovastatin and
the related compound compactin, also known as
mevinolin and mevastatin, are used to lower
serum cholesterol (Endo et al. 1976; Alberts et al.
1980). Compactin produced by Penicillium spe-
cies differs from lovastatin only in the absence of
the C-12 methyl group (Brown et al. 1976). Both
compounds efficiently inhibit mammalian hydro-
xymethylglutaryl (HMG)‐CoA reductase. Another
potent pharmaceutical against elevated serum
cholesterol is squalestatin from Phoma sp. and
other filamentous fungi (Dawson et al. 1992).
Squalestatin is an inhibitor of mammalian squa-
lene synthase and shows curative activity against
prion-infected neurons (Bate et al. 2004).
III. Biosynthesis of Polyketides
Polyketide biosynthesis is closely related to fatty
acid biosynthesis (Birch and Donovan 1953). Iso-
tope labelling experiments showed that repetitive
Claisen condensations of an acyl-coenzyme A
(CoA) starter unit with malonyl-CoA elongation
units form the carbon backbone of fatty acids and
polyketides (Fig. 16.3). Fatty acid synthases (FAS)
and PKS contain an obligatory set of ketosynthase
(KS), acyltransferase (AT) and acyl carrier protein
(ACP) domains. A conserved serine of the apo-
ACP needs to be post-translational modified with
a CoA-derived 4-phosphopantetheine (PP) arm
that is transferred by means of a PP transferase
(Keszenman-Pereyra et al. 2003). The ACP
domains serve as an anchor for both the malonyl
extender units and transiently the growing acyl
chain. The Claisen condensation is catalysed by
the KS while the AT transfers acyl groups from
OH OH
Melanin
OH
1,8-DHN
334 Julia Schuemann and Christian Hertweck
KS AT ACP
- CO2
S S
O O Claisen
R condensation
O O
OH
Enz Enz
S ER S DH
O O
R R
Fig. 16.3. Basic mechanisms of fungal polyketide biosynthesis
CoA onto the KS and ACP domains. While FAS
reduce every newly formed b-ketothioester to a
saturated chain while attached to the ACP, there is
greater flexibility in the PKS systems. Consequently,
fungal PKS can be classified according to the degree
of reduction (or more exactly b-keto processing) of
the products into non-reducing, partially or highly
reducing PKS. Several additional domains are
involved in b-keto processing: First a b-ketoreduc-
tase (KR) reduces the keto group to a secondary
alcohol. Then elimination by a dehydratase (DH)
leads to an unsaturated thioester before an enoyl
reductase (ER) produces a saturated ester.
As soon as the polyketide chain has reached
its final length, a thioesterase (TE) hydrolyses the
thioester and releases the carbon chain from
the enzyme. Notably, TE domains are not found
in every PKS. Fungal PKS C-terminal domains can
also have cyclisation activity and release products
through intramolecular ring formation (Fujii et al.
2001).
Fungal PKS can possess another intrinsic
domain for C-methyl transfer (CMeT). Unlike O-
and N-methylations, which are performed by
“post-PKS” enzymes after polyketide assembly,
fungal methylation of the polyketide carbon
backbone takes place during chain formation
(Hutchinson et al. 2000; Nicholson et al. 2001).
Although the multidomain enzymes usually
act in an iterative fashion, interestingly the degree
of reduction can vary in each extended unit.
b-Keto processing domains KR, DH, ER are
optionally used in every extension round and
therefore lead to a large variability in polyketides
not only in chain length (Staunton and Weissman
O O O
KS AT ACP
R S Enz
n
SH a S
O
(poly)phenolics
R
b
Enz
S KR
OH O
O
R S Enz
HO partially or highly
R b-keto procession reduced polyketides
2001; Cox 2007). The factors governing this varia-
bility are largely unknown.
IV. Fungal Polyketide Synthase Classes
Polyketide synthases are classified according to
their protein architecture and way of action. In
analogy to multidomain type I FAS, type I PKS are
large enzymes that contain all necessary enzymatic
activities on a single protein. In contrast, type II
PKS form a complex of single proteins with sepa-
rated enzyme activities. While bacteria possess
both non-iterative type I PKS containing indi-
vidual modules with all necessary domains for
every single elongation step and iterative type II
PKS, in fungi mainly iterative type I PKS were
found. They repeatedly use their enzymatic activi-
ties to elongate the growing acyl ester. An excep-
tion are lovastatin diketide synthase (LDKS) from
A. terreus (Kennedy et al. 1999) and compactin
diketide synthase (CDKS) from Penicillium citri-
num (Abe et al. 2002). These type I PKS catalyse
only a single elongation to synthesise a reduced
diketide product. Type III PKS were found
in plants, bacteria and fungi. They are simple KS
proteins producing chalcones, stilbenes and small
aromatic compounds (Staunton and Weissman
2001; Seshime et al. 2005).
As mentioned above, fungal type I PKS are
further classified according to the degree of reduc-
tion of their products into non-reducing (NR)
PKS, partially reducing (PR) PKS and highly
reducing (HR) PKS (Bingle et al. 1999; Nicholson
et al. 2001). A particular type of HR PKS is a
 Biosynthesis of Fungal Polyketides
hybrid synthetase composed of a HR-PKS that is
fused to a non-ribosomal peptide synthetase
(NRPS) module. These PKS-NRPS systems pro-
duce polyketides that are fused to an amino acid
by an amide bond (Song et al. 2004).
Phylogenetic studies have shown that the pro-
gramming of polyketide assembly and degree of
reduction is reflected in the KS gene and deduced
amino acid sequences (Kroken et al. 2003). Bingle
et al. (1999) designed degenerated primers based
on conserved DNA sequences of KS domains to
selectively amplify fragments of the fungal NR
type (WA type) and PR PKS (MSAS type). In a
subsequent study Nicolson et al. (2001) comple-
mented this set with selective degenerated primers
targeting KS domains of HR PKS (lovastatin type),
KR domains and CMeT domains, respectively.
These genetic tools were successfully used to
identify various fungal PKS genes like citrinin
synthase from M. purpureus (Shimizu et al.
2005), squalestatin synthase from Phoma sp.
C2932 (Cox et al. 2004) and chaetoglobosin syn-
thetase from P. expansum (Schuemann and
Hertweck 2004).
A. Non-Reducing PKS
Orsellinic acid is the simplest known fungal poly-
ketide because it is formed through the cyclocon-
densation of a non-reduced tetraketide. Four
decades ago it was shown that a cell-free extract
from Penicillium madriti is capable of producing
orsellinic acid in vitro, thus representing the first
observed PKS activity (Gaucher and Shepherd
1968). However, the nature of the PKS is
still unknown. NR PKS generally lack domains
for b-keto processing. They possess N-terminal
starter unit acyl transferase (SAT) domains, re-
sponsible for loading a starter unit, followed by
the typical KS, AT and ACP domains necessary for
chain elongation (Crawford et al. 2006). Another
conserved region has been identified between AT
and ACP, named product template (PT) domain.
Crawford et al. (2008) showed that this domain is
involved in correct cyclisation chemistry of ACP-
bound polyketides and increased product release
from the enzyme. Some known NR PKS feature
additional processing activities like cyclase
(Watanabe et al. 1998), methyl transferase
(Shimizu et al. 2005), reductase domains (Cox
2007) or additional ACPs (Fujii et al. 2001).
335
Several NR PKS genes have been characterised
that are involved in the biosynthesis of norsolorinic
acid in A. parasiticus (Minto and Townsend 1997),
A. nidulans (Minto and Townsend 1997) and
Dothistroma septosporum (Bradshaw et al. 2006)
and in the formation of pigment precursors like
YWA1 from A. nidulans (Watanabe et al. 1999) and
A. fumigatus (Watanabe et al. 2000). Another im-
portant example is tetrahydroxyl naphthalene
(THN) synthase from C. lagenarium (Takano
et al. 1995) and W. dermatitidis (Feng et al. 2001).
Labelling experiments revealed that some fun-
gal NR PKS can use short-chain fatty acids as
starter units instead of acetyl-CoA (Minto and
Townsend 1997; Kroken et al. 2003). Norsoloronic
acid, a precursor of aflatoxin, is biosynthesised
from a hexanoate starter unit. Two genes of the
aflatoxin biosynthesis gene cluster from A. para-
siticus, fas-1 and fas-2, are related to fungal FAS
genes and were shown to be involved in hexanoate
synthesis (Hitchman et al. 2001; Watanabe and
Townsend 2002). Zearalenone biosynthesis in
Gibberella zeae requires two PKS genes that code
for one HR and one NR PKS (Gaffoor and Trail
2006). The HR PKS (ZEA2p) likely generates a
hexaketide starter unit that is loaded onto the
NR PKS (ZEA1p) SAT domain and then under-
goes three further elongations (Fig. 16.4).
The PT domain is a yet little understood region
of about 350 residues. Sequences of known PT
domain regions group according to the chain length
of their products. This suggests that they have a
similar function in the determination of extension
number like the KSb (or chain length factor, CLF)
component of bacterial type II PKS (Keatinge-Clay
et al. 2004; Cox 2007). Deconstruction experiments
by domain dissection and reassembly of the
A. parasiticus PksA revealed the function of PT in
product formation (Crawford et al. 2008). During
aflatoxin biosynthesis PT was shown to drive aro-
matisation chemistry to irreversibly form rings A
and B and to increase the flux of the norsoloronic
acid precursor from the PKS enzyme.
TE domains are the most common N-terminal
processing domains found in NR PKS. They can
operate as thioesterases or as Claisen cyclases
(CYC) as shown by Ebizuka and others (Watanabe
et al. 1998; Watanabe et al. 1999; Fig. 16.5). Heter-
ologous expression of the A. nidulans YWA1
synthase (WAS) gene wA in full-length and
truncated variants thereof showed that the CYC/
TE is not necessarily required for the release of the
336 Julia Schuemann and Christian Hertweck

Fig. 16.4. Biosynthesis of zeara- 1 Acetyl-CoA

lenone by NR-PKS ZEA1p and 5 Malony-CoA
HR-PKS ZEA2p

OH O O
ZEA2p
S

3 Malonyl-CoA

OH O O O O O
ZEA1p
S

OH O

O
ZEA1p
O S

O O

O O HO
ZEA1p
S

OH O

HO

O
Zearalenone

polyketide product. Nonetheless, a conserved his-
tidine and a serine residue are crucial for CYC/TE
activity (Fujii et al. 2001). A similar CYC/TE do-
main occurs in A. nidulans sterigmatocystin
synthase PKSst (Yu and Leonard 1995).
Another function of C-terminal TE domains is
the determination of chain length. C. aegenarium
PKS1 naturally produces the pentaketide THN
(Takano et al. 1995). Mutants of the PKS1 TE
domain resulted in production of mainly a
hexaketide product showing that the deletion of
TE or mutation of conserved residues influences
the polyketide chain length (Takano et al. 1995;
Watanabe and Ebizuka 2004).
Some fungal NR PKS contain multiple ACP
domains. A. nidulans WAS (Watanabe et al.
1999), NSAS (Minto and Townsend 1997) and
C. lagenarium THNS (Takano et al. 1995) contain
two ACPs and D. septosporum NSAS even three
copies (Bradshaw et al. 2006). The additional
 Biosynthesis of Fungal Polyketides
CLC
SAT KS AT PT ACP WAS
/TE HO
S S O
O HO O
OO
HO
HO
6 Malonyl-CoA
ACPs are not required for biosynthesis, but all
ACP domains are functional and exchangeable
even among different species (Fujii et al. 2001;
Watanabe and Ebizuka 2002).
Kroken et al. (2003) identified a group of
NR PKS containing a CMeT domain downstream
of ACP. The only known sequence that is corre-
lated with a secondary metabolite is the citrinin
synthase (CitS) gene from M. pupureus. CitS
likely elongates a reduced diketide starter unit to
citrinin (Shimizu et al. 2005). In this case CMeT
must act twice but it is not clear how the point
in time for methylation is programmed (Hajjaj
et al. 1999).
Sequence analyses show that M. pupureus CitS
possesses a C-terminal thioester reductase. In a
mechanism similar to NRPS systems, the CitS
reductase domain might be involved in the release
of the product as an aldehyde or primary alcohol
(Shimizu et al. 2005; Cox 2007).
B. Partially Reducing PKS
Fungal PR PKS differ from NR PKS in that they
contain an N-terminal KS, followed by AT, DH, a
core domain (core), KR and a C-terminal ACP.
There are no SAT, PT or TE domains. Only three
fungal genes coding for PR PKS have been
matched to secondary metabolites, although
many more fungal PR PKS genes are known.
These three studied genes code for fungal
6-MSAS (Fig. 16.6). One was isolated from
P. patulum (MSAS; Beck et al. 1990), one from
A. terreus (atX; Fujii et al. 1996) and another
337
Fig. 16.5. Structure of WAS and
OO biosynthesis model
OH O O
O
OH
OH OH O
YWA1
gene (pks2) was discovered in Glarea lozoyensis
(Lu et al. 2005).
P. patulum and A. terreus MSAS are both
supposed to form homotetramers. Fujii and co-
workers studied the recombination of functional
MSAS complexes from deletion mutants of the
A. terreus atX gene (Moriguchi et al. 2006). They
expressed two copies of atX in S. cerevisiae simul-
taneously. This allowed them to mutate indepen-
dently and to cross-complement mutants. Almost
all combinations of truncated MSAS proteins were
functional, showing that all truncations can be
complemented by another functional copy of
the protein. Only one short 122-amino-acid region
of the core domain was necessary for comple-
mentation. Probably this region codes for a
motif required for subunit–subunit interaction.
A similar core sequence was found in other fungal
PR PKS and in bacterial PKS. Also, mammalian
FAS possess a core region with similar function
in enzyme assembly (Witkowski et al. 2004).
However, no sequence similarities were found
between fungal MSAS and mammalian FAS core
regions.
C. Highly Reducing PKS
HR PKS differ from NR and PR PKS by an extended
set of domains. The N-terminal KS domain
is followed by AT, DH, ER, KR domains, and a C-
terminal ACP domain. In some cases, however,
the protein lacks a conserved ER domain, in lieu
harbouring a region without known function.
These PKS apparently utilize a free-standing
ER in trans. In addition to the full set of b-keto
338 Julia Schuemann and Christian Hertweck
Fig. 16.6. Architecture of the
MSAS and model for the biosyn- KS AT DH Core KR ACP
thesis of 6-MSA
S S
O O
3 Malonyl-CoA
3 SAM ()
processing domains, many HR PKS possess a
CMeT domain following a DH.
Genes linked to known highly reduced meta-
bolites are involved in the biosynthesis of lovastat-
in produced by A. terreus (Kennedy et al. 1999),
compactin from P. citrinum (Abe et al. 2002), zear-
alenone from G. zeae (Gaffoor and Trail 2006),
squalestatin produced by Phoma sp. (Cox et al.
2004), T-toxin from C. heterostrophus (Yang et al.
1996; Baker et al. 2006) and fumonisin from G.
fujikuroi (Proctor et al. 1999).
Lovastatin and compactin are both biosyn-
thesised by two separate PKS. Genes lovB and
lovF (Hendrickson et al. 1999; Kennedy et al.
1999) encode lovastatin nonaketide synthase
(LNKS) and lovastatin diketide synthase (LDKS),
respectively, that are involved in lovastatin
biosynthesis (Fig. 16.7). Compactin nonaketide
synthase (CNKS; encoded by mlcA) and compac-
tin diketide synthase (CDKS; encoded by mlcB)
assemble compactin (Abe et al. 2002). Isotope
feeding experiments suggested the synthesis of a
reduced nonaketide chain that would undergo a
Diels–Alder reaction to form dihydromonacolin L
(Moore et al. 1985; Witter and Vederas 1996).
LNKS has an inactive ER domain and requires
the assistance of the separately encoded ER LovC
for correct function. The involvement of a trans-
acting ER was supposed for CNKS as well.
Surprisingly, LNKS possesses a C-terminal trun-
cated condensation (C) domain typical for NRPS.
It is not clear whether this domain has a function
in offloading or if it is just a relict of truncation.
LDKS is a HR PKS with KS, AT, CMeT, ER, KR
and ACP domains. Notably, this is not an iterative
PKS; in order to synthesise a diketide only a single
elongation step is required. Acyl transferase LovD
6-MSAS
O OH
HO
O
OH 6-MSA
transfers the diketide onto C-10 hydroxyl of mon-
acolin J to assemble lovastatin (Kennedy et al.
1999). Xie et al. (2006) showed that LovD, pro-
duced in E. coli, can transfer various acyl-CoA
residues onto monacolin J.
Squalestatin tetraketide synthase (SQTKS)
was cloned from Phoma sp. (Cox et al. 2004). It
shows high similarity to LDKS but produces a
tetraketide chain. Squalestatin is assembled from
two methylated polyketide chains, a hexaketide
and a tetraketide chain. Notably, the hexaketide
chain is synthesised from an unusual benzoate
starter unit (Jones et al. 1992).
T-toxin is a very long, linear C41 polyketide
produced by the maize pathogen C. heterostro-
phus (Kono and Daly 1979). It was one of the
first compounds linked to a defined PKS gene
(Yang et al. 1996). Later it turned out that two,
not clustered genes are involved in T-toxin bio-
synthesis (Baker et al. 2006). Genes pks1 and pks2
code for T-toxin synthases TTS1 and TTS2, which
both represent typical HR PKS. Interestingly,
TTS1 possesses a CMeT domain although
T-toxin does not show any methyl branches.
The mycotoxin fumonisin B1 causes several
human and animal diseases (Nelson et al. 1993). It
contains a methylated nonaketide chain, esterified
to two unusual C7 tricarboxylic acids. Fumonisin
synthase (FUMS) appears to be a typical HR PKS
with a functional ER domain. It is encoded by
fum1 in G. fujikuroi (Proctor et al. 1999).
The plant pathogen Alternaria solani pro-
duces numerous polyketides, for example solano-
pyrone A and alternaric acid (Fig. 16.8). PCR
screening with primers for conserved PKS
sequences led to the identification of two PKS
genes (Fujii et al. 2005). One gene (alt5) codes
 Biosynthesis of Fungal Polyketides 339

HO
COOH
KS AT DH CMeT KR ACP C LovB + Lov C
OH

S S
O
ER LovC O Lov B only

OH OH
8 Malonyl-CoA
SAM ( ) OH

O O enzymatic
Diels–Alder

Heptaketide pyrone
HO O
OH
O

O O
H

Hexaketide pyrone
H
Dihydromonacolin L

KS AT DH CMeT ER KR ACP LovF

S S
O O
HO O
HO O OH
O
O Malonyl-CoA
O SAM ( )
OH
O H
H
Lov D

Monacolin J
Lovastatin

Fig. 16.7. Lovastatin biosynthesis involving LovB and LovF

for a HR PKS (PksN) and another (pksA) for a NR
PKS. Heterologous production of PksN in Asper-
gillus oryzae led to the production of the methy-
lated decaketide alternapyrone. Two more HR
PKS sequences (pksF, pksK) were identified
through gene probing of the Aspergillus solani
genome. Both PKS genes lack a CMeT region
(Kasahara et al. 2006). PksK was inactive when
expressed in A. oryzae. However, the heterologous
expression of PksF produced two mayor com-
pounds, aslanipyrone and aslaniol. Both are poly-
unsaturated suggesting that the ER domain is
inactive.
D. Polyketide Synthase–Non-Ribosomal Peptide
Synthetase Hybrids
Many structurally intriguing fungal metabolites
with remarkable biological activities feature a poly-
ketide backbone fused to an amino acid. The bio-
synthesis of such hybrid metabolites is well studied
in bacteria, e.g. biosynthesis of the siderophore and
virulence factor yersiniabactin from Yersinia pestis
(Miller et al. 2002). Fungal PKS-NRPS hybrids have
a highly reducing PKS region containing an N-
terminal KS, AT, DH, CMeT, inactive ER, KR and
ACP domains. The ACP is flanked by domains of a
340 Julia Schuemann and Christian Hertweck
CHO
O OMe
HO
O equisetin has been examined by Schmidt and col-
OH O leagues (Sims et al. 2005). Equisetin synthetase
HO COOH
Solanayrone Alternaric acid
OH
O O
Alternapyrone
O (2004) could further show that production of the yet
O OH
HO
Aslanipyrone
OH
Aslaniol
Fig. 16.8. Compounds produced by Alternaria solani PKS
typical NRPS module: condensation (C), adenyla-
tion (A) and peptidyl carrier protein (PCP)
domains. Furthermore, the hybrid systems feature
an additional C-terminal domain that might either
function as a thioester reductase (R) or cyclase
domain (Song et al. 2004). Like in lovastatin bio-
synthesis, a trans ER may be required for the cor-
rect PKS function (Halo et al. 2008).
PKS-NRPS hybrid synthetases are involved in
the biosynthesis of fusarin C in Fusarium monili-
forme (Song et al. 2004), equisetin from F. hetero-
sporum (Sims et al. 2005), tenellin produced in
Beauveria bassiana (Eley et al. 2007), aspyridone
from A. nidulans (Bergmann et al. 2007), chaeto-
globosin produced by P. expansum (Schuemann
and Hertweck 2004) and pseurotin A from
A. fumigatus (Maiya et al. 2007) (Fig. 16.9).
Biosynthesis of the mycotoxin fusarin C starts
with the assembly of a tetramethylated heptake-
tide that is fused to homoserin (Song et al. 2004).
The reductive release of the hybrid product
and subsequent cyclisation putatively forms
prefusarin, which is then transformed into fusarin
O
C by various tailoring enzymes.
O
Biosynthesis of the HIV-1 integrase inhibitor
(EqiS) has the same overall domain architecture as
fusarin synthetase (FUSS). It differs from FUSS in
the formation of a methylated oktaketide chain that
is linked to serine. Another gene cluster containing
a PKS-NRPS gene was identified within the genome
of the plant pathogen M. grisea (Böhnert et al.
2004). The product of the encoded hybrid enzyme
is yet unknown. It was shown that the gene cluster
is expressed only during appressorium formation
during penetration of the rice plant. Böhnert et al.
unknown fungal compound leads to resistance of
the rice plant to further fungal penetration.
The yellow pigment tenellin from the insect
pathogen B. bassiana is produced by the PKS-
NRPS TENS (Eley et al. 2007). A dimethylated
OH
pentaketide fused to tyrosine is synthesised
to build pre-tenellin after reductive release. Tai-
loring reactions performed by two putative P450
monooxygenases and rearrangements probably
convert the precursor into tenellin. The hypothe-
sis that tenellin might be involved in the pathoge-
nicity of B. bassiana was ruled out by the fact that
a direct knockout of TENS did not significantly
reduce pathogenicity towards wax moth larvae.
A genome mining approach identified the
only gene cluster of the A. nidulans genome con-
taining a silent PKS-NRPS hybrid gene (Bergmann
et al. 2007). Promoter exchange of the only clus-
tered regulator gene led to the production of novel
polyketide-amino acid hybrid compounds
(Fig. 16.10). Aspyridone synthetase ApdA likely
produces a dimethylated tetraketide and fuses it
to a tyrosine moiety. A reductive downloading
mechanism might release an amino aldehyde
that could undergo cyclisation to a pyrrolinone.
The involvement of oxidative tailoring reactions
towards tetramic acid intermediates and ring rear-
rangements probably form the moderately cyto-
toxic aspyridones A and B. Aspyridone
biosynthesis seems to be closely related to tenellin
biosynthesis reported by Eley et al. (2007).
Cytochalasin (chaetoglobosin) biosynthesis
was investigated in the fungus P. expansum
(Schuemann and Hertweck 2004). Screening a
genomic library of P. expansum led to the
 Biosynthesis of Fungal Polyketides 341

O Fig. 16.9. PKS-NRPS hybrid me-

N H tabolites
OH
O
H O
O OH

N O O
H HO
H
Equisetin Fusarin C

HO HO
OH O OH O

N O N O
H
OH
Tenellin Aspyridone A: R = H
Aspyridone B: R = OH

H
OH O
HN
O NH O
22
N O O OH
21
H OCH3
R O OH
O
Chaetoglobosin A: R = OH, C21-C22 Pseurotin A
Chaetoglobosin C: R = O, C21=C22

identification of a gene cluster encoding a PKS-
NRPS (CheA; Fig. 16.11). A RNA-silencing meth-
od proved that CheA is involved in chaetoglobosin
production in P. expansum. It was suggested that
CheA produces a trimethylated nonaketide that is
then fused to a tryptophan moiety (Fig. 16.12).
After reductive release of the hybrid product, cat-
alysed by the C-terminal R domain, cyclisation to
a pyrrolinone product likely occurs. This would
act as a dienophile and undergo a Diels–Alder
reaction with the diene moiety of the polyketide
chain. Tailoring reactions seem to be carried out
by the putative cytochrome P450 monooxy-
genases CheD and CheG and by the encoded
FAD-dependent monooxygenase CheE to yield
chaetoglobosin A and C. A Diels–Alder-like cycli-
sation mechanism was also proposed for lovastat-
in and equisetin biosynthesis (Oikawa and
Tokiwano 2004; Sims et al. 2005). Oikawa and
Tokiwano proposed the involvement of enzymatic
Diels–Alderases for cyclisation of natural product
precursors, e.g. polyketides. It is conceivable that
PKS–NRPS synthetases enzymatically catalyse this
reaction under physiological conditions or that they
act as an entropic trap.
A single PKS-NRPS hybrid gene was identified
within the sequenced genome of A. fumigatus.
Deletion of the hybrid gene named psoA in
A. fumigatus led to the absence of pseurotin A,
while overexpression led to accumulation of the
hybrid metabolite (Maiya et al. 2007). Pseurotin A
is a compound with unusual heterospirocyclic
gamma-lactame structure and is a competitive
inhibitor of chitin synthase and inducer of nerve-
cell proliferation (Bloch et al. 1976; Komagata
et al. 1996).
V. Post-PKS Modifications
The large structural diversity of polyketides
results not only from varying chain lengths, de-
gree of b-keto processing and cyclisation, but also
from post-PKS modifications of the carbon chain
by alkylation, acylation or oxygenation.
An impressive set of post-PKS modifications
is involved in aflatoxin biosynthesis (Fig. 16.13).
Aflatoxins, produced by some Aspergillus species,
are a group of structurally related polyketide-
derived furanocoumarins. In a 70-kb gene cluster
at least 25 genes are involved in a complex biosyn-
thetic pathway. Beside the PKS gene (pksA) and
two FAS genes (fas-1, fas-2) they code for cyto-
chrome P450 monooxygenases, dehydrogenases,
methyltransferases, pathway regulators and a
342 Julia Schuemann and Christian Hertweck
apdB apdC apdD apdE apdA
ectopic integration of regulator gene
apdB apdC apdD apdE apdA
PKS-NRPS
tailoring
regulatory genes
transporter
P inducible promoter
Fig. 16.10. Activation of the silent aspyridone gene cluster via ectopic integration of inducible regulator copy
transporter. Fifteen of those genes catalyse oxida-
tive reactions. Most of the clustered genes are
regulated by a single Zn2Cys6-type transcription
factor AflR and by the co-regulator AfkJ. In total
about 21 enzymatic steps are required for aflatox-
in biosynthesis (Minto and Townsend 1997;
Bhatnagar et al. 2003).
Treatment of fungal cultures with inhibitors
of post-PKS enzymes can significantly affect the
product spectrum. For example, Chaetomium sub-
affine, producer of chaetoglobosin A, was incu-
bated with the cytochrome P450 inhibitor
metyrapone (Oikawa et al. 1991). Chaetoglobosin
A production was reduced while accumulation of
less oxidised precursor compounds was observed,
thus providing an insight into the final biosyn-
thetic steps.
In post-PKS reactions polyketides can also
be fused to further polyketide chains. Acyl trans-
ferase Lov D transfers a methylbuturyl side chain
onto the lovastatin precursor monacolin J in order
to fully assemble lovastatin (Hendrickson et al.
1999; Kennedy et al. 1999).
VI. Phylogeny
In an extensive phylogenetic study Kroken et al.
(2003) compared the amino acid sequences of
mainly fungal KS domains. Fungal type I PKS
sequences form a major clade together with bac-
terial type I PKS. This clade is clearly sister to
animal FAS and also bacterial type II PKS. Type I
apdF Reg apdG
P
apdF Reg apdG Reg
P
HO
OH O
R
N O
H
Aspyridone A: R = H
Aspyridone B: R = OH
PKS fall into three main clades; two of these
contain exclusively fungal sequences and corre-
late with their products reduction: reducing PKS
and non-reducing PKS. They are mainly charac-
terised by the presence or absence of b-keto pro-
cessing domains (DH, ER, KR). Each main fungal
clade is further divided into four groups with
characteristic domain sets resulting from loss
of ancestral domains or from gain of novel ones
(e.g. NRPS domains). The ancestral type I PKS
domain structure is supposed to be: KS, AT, DH,
CMeT, ER, KR, ACP. The first main fungal clade
contains PKS that are involved in the production
of reduced, mainly linear polyketides like
precursors of toxins (e.g. lovastatin, fumonisin,
T-toxin). The second main fungal clade includes
PKS that synthesise unreduced, cyclic polyke-
tides, such as toxins (e.g. aflatoxin) and pigments
(e.g. melanin). Two more fungal clades were
found both nested into the bacterial type I PKS
clade. One contains 6-MSA-type PKS. The
other consists of a single sequence, the C. hetero-
strophus PKS24 probably a PKS-NRPS hybrid.
Orthologous PKS genes, producing nearly identi-
cal polyketides, were found among closely related
but also distant species. Due to the diversity and
distribution of fungal PKS it was speculated that
gene duplication, divergence and differential gene
loss are responsible for generation and mainte-
nance of this diversity. Horizontal gene transfer is
not necessarily involved (Kroken et al. 2003).
In contrast, horizontal gene transfer was pro-
posed as one way for the evolution of virulence
 Biosynthesis of Fungal Polyketides 343

cheC cheB cheA cheD cheE cheF cheG

2 kb
* PKS-NRPS
tailoring
mRNA (cheA) regulatory genes
*
* Chaetoglobosins
CheA

mRNA (cheA)

antisense mRNA
X CheA

inverted
alcAp cheA fragment

Fig. 16.11. Organisation of the chaetoglobosins gene cluster. (a) P. expansum wild type producing chaetoglobosins (*)
and (b) silencing mutant with reduced chaetoglobosin synthesis

KS AT DH CMeT KR ACP C A PCP R CheA

S S S
O ER CheB O O NAD(P)H

O HN O
O H
8 Malonyl-CoA N N H O
3 SAM ( ) H R
O
R N O
H

R - H2O

O
HN
O
N
H
R

Trp-AMP

H H
CheDEG
HN 22
HN
N O O N O O
21
H H
R R
O
Chaetoglobosin A: R = OH, C21-C22
Chaetoglobosin C: R = O, C21=C22

Fig. 16.12. Architecture of the chaetoglobosin synthetase CheA and model for biosynthesis
344 Julia Schuemann and Christian Hertweck

norB cypA aflT pksA nor-1 fas-2 fas-1 aflR aflJ

37 kb

estA ver-1
adhA norA verA avnA verB avfA omtB omtA ordA vbs cypX moxYordB hypA
70 kb

polyketide biosynthesis fatty acid synthase

tailoring unknown
regulatory genes transporter

O O
FAS
EnzS CoAS
PKS

O O SEnz OH O OH
O OH O OH O
O PKS O
O O
HO O
O O HO OH
O
O
Norsolorinic acid Averufin

O O OH O OH
O HO O OH
O
O O O HO O
steps
MeO O absent in O O
MeO
A. nidulans
Aflatoxin B1 Sterigmatocystin Versicolorin B

Fig. 16.13. Aflatoxin biosynthetic gene cluster and biosynthesis model according to Bhatnagar et al. (2003)

genes in filamentous fungi, e.g. the PKS-NRPS They clustered in two clades of Schmitt’s align-
encoding gene ACEI from M. grisea (Böhnert ment. Amino acid sequence of KS domains from
et al. 2004). While phylogenetic analyses reveal 15 Leconora species, three other lichen species
complex duplication events for the evolution of and other non-lichenised fungi were compared.
ACEI-like clusters in Chaetomium globosum and Leconora PKS sequences clustered mainly with
M. grisea, horizontal gene transfer is proposed PKS producing complex aromatic compounds,
from a relative of M. grisea into an ancestor of with non-lichen-derived PKS producing precur-
Aspergillus clavatus (Khaldi et al. 2008). sors of DHN-melanins and in another clade
Additional phylogenetic analysis of the hybrid without relationship to known fungal PKS.
synthetase EqiS indicate that fungal PKS-NRPS
have likely diversified by point mutation and
deletion (Sims et al. 2005). This is in contrast to VII. Functional Analysis
bacterial PKS systems, which use module and
domain swaps to generate structural diversity
and Engineering of Fungal
(Crosa and Walsh 2002). Polyketide Biosynthesis
Phylogenetic studies were also carried out on
KS sequences of NR PKS from Pertusariales Engineering fungal polyketide biosynthesis genes
(lichenised fungi) and other NR PKS sequences or gene clusters could be an efficient way to diver-
(Schmitt et al. 2005). In alignments the sequences sify metabolite structures and their biological
grouped in 18 clades containing only lichenised activities (Burkart 2003; Reeves 2003; Schuemann
taxa, only non-lichenised taxa or sequences from and Hertweck 2006).
both. Interestingly, clades could not be associated From the gene loci that have been investigated
with secondary metabolism classes. Another study it appears that nearly all genes necessary for fungal
concerned sequence comparison of mainly Leco- polyketide biosynthesis, post-PKS processing, regu-
nora spp. PKS sequences (Grube and Blaha 2003). lation and resistance are clustered, as exemplified
 Biosynthesis of Fungal Polyketides
for the lovastatin (Kennedy et al. 1999) and aflatoxin
biosynthesis gene clusters (Yu et al. 1995; Bhatnagar
et al. 2003; Fig. 16.13). This organisation is an adva-
ntage for identification and handling of gene cas-
settes or even whole gene clusters.
Pathway engineering towards novel com-
pounds has been intensely explored in bacteria
(Cane et al. 1998; Walsh 2002; Weber et al. 2003),
but in fungi such combinatorial biosynthesis
approaches have been far more challenging from
a biotechnical point of view. The incidence of
introns in fungal genes can be problematic for
heterologous expressions and also the directed
integration of DNA into fungal genomes is not
guaranteed and may be cumbersome.
A. Inactivation of PKS Genes
1. Knockout
The disruption or replacement of PKS genes is a
common approach for their functional analyses.
However, the efficiency of targeting a genetic
locus can vary significantly between fungal spe-
cies. Apart from challenges regarding selection
markers and introduction of DNA into the fungus,
the integration of transformed DNA can occur in a
homologous or non-homologous manner.
The lovastatin biosynthesis genes were identi-
fied by screening mutants of the producer strain
A. terreus in 1999 (Hendrickson et al. 1999;
Kennedy et al. 1999). Several mutants were inves-
tigated that were unable to produce lovastatin or
that yielded polyketide shunt products only, which
wild type
ORF1 ORF2 fusA
knock out mutant
ORF1 ORF2 fusA
hph
PKS-NRPS
tailoring
transporter
unknown
hph hygromycin-resistance cassette
Fig. 16.14. Gene knockout of the F. moniliforme gene fusA
345
led to the discovery of the lovastatin biosynthesis
gene cluster including both PKS genes lovB and
lovF (Fig. 16.7). Further directed gene disruptions
proved the involvement of lovC and lovD in lova-
statin biosynthesis (Kennedy et al. 1999). Similar
knockout experiments in P. citrinum correlated
gene mlcA with compactin production (Abe et al.
2002).
Targeted gene knockout experiments also
revealed the function of PksA from A. parasiticus
(Chang et al. 1995). The corresponding gene pksA
is similar to the A. nidulans wA gene and converts
hexanoyl-CoA into norsolonic acid, an intermedi-
ate in sterigmatocystin and aflatoxin biosynthesis
(Trail et al. 1995; Brown et al. 1996; Bennett et al.
1997; Minto and Townsend 1997; Bhatnagar et al.
2003).
Directed gene replacement proved the function
of pks1, a gene coding for a PKS involved in DHN-
melanin synthesis in G. lozoyensis. Knockout
mutants were achieved by double crossover recom-
bination via Agrobacterium-mediated transforma-
tion (Zhang et al. 2003).
Directed knockout experiments in F. monili-
forme and F. venenatum correlated a PKS-NRPS
hybrid gene with fusarin C biosynthesis (Song
et al. 2004; Fig. 16.14). Likewise, a gene knockout
by homologous recombination in F. heterosporum
revealed that equisetin biosynthesis requires the
gene product of pks2 (Sims et al. 2005).
In 2007 the PKS-NRPS hybrid gene psoA was
associated with pseurotin A production in A. fumi-
gatus by deletion and overexpression (Maiya et al.
2007).
ORF4 ORF5 ORF6 O
O
OH
N O O
HO H
Fusarin C
ORF4 ORF5 ORF6
no fusarin production
346 Julia Schuemann and Christian Hertweck
The generation of random mutations through
restriction enzyme-mediated integration (REMI;
Brown et al. 1998), UV-mutagenesis (Jahn et al.
1997) or transposon mutagenesis (Kempken et al.
1998; Kempken and Kuck 2000; Brakhage and
Langfelder 2002) has been successfully used to
target fungal genes. The REMI procedure was
used to identify a PKS gene involved in T-toxin
production in the fungal pathogen C. heterostro-
phus (Yang et al. 1996).
2. Knockdown
Another genetic tool to study PKS gene functions
in fungi is RNA-mediated gene silencing. In this
post-transcriptional down-regulation method
double-stranded RNA triggers the degradation of a
sequence homologous mRNA. With this method
Nakayashiki et al. (2005) successfully silenced
specific genes in Magnaporthe oryzae and
C. lagenarium.
RNA silencing is an attractive method to ana-
lyse lethal knockouts or cases when directed gene
disruptions failed due to a low efficacy of homol-
ogous integration events in the fungal genome.
This method was successfully applied in the inves-
tigation of chaetoglobosin biosynthesis in
P. expansum (Schuemann and Hertweck 2004).
All efforts to disrupt the proposed PKS-NRPS
gene cheA of the sequenced gene cluster failed.
Finally, cheA RNA-silencing resulted in a signifi-
cant decrease in chaetoglobosin production,
which proved the involvement of the targeted
gene in metabolite biosynthesis (Fig. 16.11).
Sequence similar genes can be silenced simul-
taneously. A disadvantage is that silencing not
necessarily leads to a null phenotype. This may
be crucial for the down-regulation of non-major
activity enzymes (like tailoring enzymes), which
might have no detectable effect on metabolite
production level (Thierry and Vaucheret 1996;
Baulcombe 1999).
B. Heterologous Expression of PKS Genes
A general expression system for fungal PKS genes is
preferable in order to engineer metabolite biosynthe-
sis and improve yields. The use of native polyketide
producers is often hampered by a high background
of metabolites, sub-optimal growth conditions or
low production of the desired polyketide.
In contrast to bacterial genes, fungal genes
mostly contain introns that require a strain-
specific splicing mechanism. Introns are hard
to remove and impair gene expression in non-
eukaryotic systems. In addition, apo-ACP domains
need to be post-translationally 4-phosphopan-
tetheinylated, thus making co-expression of a spe-
cific PPTase essential. Beside fungal hosts (e.g.
A. nidulans), yeast (Saccharomyces cerevisiae),
bacteria (Actinomycetes) and plants (tobacco)
have proven suitable for PKS gene expression
(Pfeifer and Khosla 2001).
The first successful expression of a fungal PKS
gene in bacteria was reported in 1995 (Bedford
et al. 1995). Bedford et al. (1995) expressed the
6-MSAS gene from P. patulum in Streptomyces
coelicolor CH999. Extensive modification of the
expressed gene was necessary. The intron was
removed, a Shine–Dalgarno sequence was cloned
in the 30 region and four codons were adapted into
more frequently used ones in Streptomycetes
(Wright and Bibb 1992). The 6-MSAS gene was
expressed under control of the S. coelicolor acti-
norhodin actI promoter.
One year later the 6-MSAS encoding gene atX
from A. terreus was identified and expressed in
the fungal host A. nidulans (Fujii et al. 1996). For
successful expression the atX gene was cloned
downstream the amyB promoter of the Taka-
amylase A gene.
In 1999 the expression of the LNKS gene in
A. nidulans using the alcA promoter for expres-
sion control was reported by Kennedy et al.
(1999). Apparently LNKS alone was malfunction-
ing and synthesised polyunsaturated pyrones only
(Fig. 16.7). Enoyl reduction and ketoreduction
had not occurred and the polyketide chain was
released prematurely. Sequence analysis revealed
that the NADPH binding pocket and this for ER
activity of LNKS was dysfunctional. Co-expres-
sion of the separately encoded and trans-acting
ER LovC complemented ER activity and led to the
production of the expected dihydromonacolin L.
Squalestatin tetraketide synthase from Phoma
sp. was introduced into A. oryzae, and the trans-
formant produced detectable amounts of the tetra-
ketide side-chain of sqalestatin S1 (Cox et al. 2004).
The only successful expression of a fungal PKS
in Escherichia coli and additionally in S. cerevisiae
was reported by Kealey et al. (1998). They
co-expressed the gene coding for 6-MSAS together
with the gene encoding Bacillus subtilis PPTase Sfp
 Biosynthesis of Fungal Polyketides
in both hosts. Using suitable promoters, alcohol
dehydrogenase 2 (ADH2) promoter and T5 RNA
polymerase promoter, respectively, high produc-
tion levels were achieved in yeast (twofold higher
than in the wild type), while 6-MSA production was
just detectable in E. coli (Kealey et al. 1998).
The 6-MSAS gene from P. patulum was also
expressed in tobacco, showing that plants could
also serve for the production of fungal PKS. Inter-
estingly, the plant PPTase seemed to convert fungal
apo-ACP into the holo enzyme (Yalpani et al. 2001).
Ebizuka and co-workers demonstrated the
expression of three HR PKS genes from A. solani
in A. oryzae (Kasahara, Fujii et al. 2006). While
PksK was non-functional, production of PksN
and PksF led to the production of the octamethy-
lated decaketide alternapyrone and aslanipyrone/
aslaniol, respectively (Fujii et al. 2005; Fig. 16.8).
Halo et al. (2008) succeeded in expressing the
tenellin synthetase gene tenS in A. oryzae. Heter-
ologous expression of the PKS-NRPS hybrid
synthetase-encoding gene tenS without the sepa-
rately encoded ER gene led to the production of
acyl tetramic acids, albeit their structure elucida-
tion revealed errors in polyketide assembly.
A correctly built polyketide could be achieved by
co-expression of tenS with orf3, coding for the
trans-acting ER of the tenellin biosynthesis gene
cluster.
In 2007 the iterative, non-reducing bikaverin
PKS, PKS4 from G. fujikuroi, was expressed at
high levels in E. coli, purified and reconstituted
to in vitro activity (Ma et al. 2007). In the presence
of malonyl-CoA PKS4 synthesised the precursor
of bikaverin as main product, demonstrating the
functionality of the bacterial expressed fungal
PKS. This work provides a basis for enzymatic
in vitro synthesis of fungal polyketide compounds.
Mutations of the G. fujikuroi PKS4 C-terminal
thioesterase/Claisen cyclase (TE/CYC) domain
were performed by Ma et al. (2008). The mutant
PKS were disabled in correct cyclisation of the
nascent polyketide, proving the involvement of
TE/CYC in the folding of bikaverin. Complemen-
tation of the mutant PKS4 with a standalone TE/
CLC domain restored the regioselective cyclisa-
tion steps. In further in vitro experiments disso-
ciated bacterial tailoring enzymes were added to
the reconstituted PKS4. Addition of the S. coelico-
lor actinothodin KR led to the C9 reduced and
shortened oktaketide mutactin. The addition of
first ring aromatase/cyclase (GRIS ARO/CYC)
347
from the griseusin PKS (Zawada and Khosla
1999) and second ring cyclase OxyN (Zhang
et al. 2007) resulted in anthraquinone products.
These experiments impressively show how fungal
PKS can be complemented with trans-acting
domains and tailoring enzymes even from distinct
families of PKS.
C. PKS Pathway Regulation
For several reasons it might be attractive to regu-
late the activity of a whole biosynthesis gene clus-
ter in order to increase, decrease or temporally
control metabolite production. Methodologies to
alter fungal secondary metabolic synthesis involve
varying environmental factors such as nutrients or
temperature. The marine fungus Phomopsis aspar-
agi was treated with the F-actin inhibitor jasplaki-
nolide in order to turn on pathways (Christian
et al. 2005). The Phomopsis genus is a rich source
for secondary metabolites. Incubation of P. aspar-
agi with jasplakinolide, first isolated from the
sponge Jaspis splendens, resulted in the production
of three new biological active chaetoglobosins.
Mining the genome of A. nidulans, an orphan
PKS gene cluster was identified (Bergmann et al.
2007). Analyses revealed the presence of one PKS‐
NRPS gene, one gene encoding an ER, four
putative tailoring genes, one gene coding for an
exporter and one single regulator gene named
apdR. All efforts to cultivate this fungus under
conditions that lead to the production of a poly-
ketide-non-ribosomal peptide related product
failed. The gene cluster remained silent under
test conditions. A novel activation method was
established in order to identify the encoded natu-
ral product biosynthesis pathway. A copy of the
regulator gene apdR, coding for a C6 transcription
factor, was cloned and expressed under control of
the inducible alcA promoter from A. nidulans
(Fig. 16.10). Induction of the regulator induced
the transcription of all biosynthesis genes and
finally led to the production of new polyketide-
non-ribosomal peptide products in A. nidulans.
New members of pyridone alkaloids were isolated,
the aspyridones.
VIII. Conclusions
Fungal PKS are highly specialised programmed
nanomachines. With a simple set of active
348 Julia Schuemann and Christian Hertweck
domains some of the most complex natural pro-
ducts are synthesised. Fungal PKS use their enzy-
matic domains iteratively, and it is not yet
thoroughly understood how chain length, degree
of reduction and C-methylation pattern are pro-
grammed. During the past years remarkable prog-
ress was made in the genetic engineering of fungal
PKS. Modifications of PKS genes, tailoring genes
or whole biosynthesis pathways were demon-
strated. Expression in heterologous hosts was rea-
lised and pilot experiments in fungal PKS domain
swaps were made. An enormous biosynthetic
potential holds the combination of polyketide bio-
synthesis with other pathways, for example non-
ribosomal peptide biosynthesis. Nature combines
those pathways in mixed biosynthetic enzymes,
such as PKS-NRPS hybrids.
Still we are far away from the status quo of
understanding and handling bacterial PKS. To
take advantage of the biosynthetic potential of
fungal PKS we have to learn much more. Future
insights in the structure and action of fungal PKS
will set the basis to identify and synthesise new
pharmaceutical active products.
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17 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis

ROLF GEISEN1, MARKUS SCHMIDT-HEYDT1

CONTENTS plant-pathogenic Fusarium species, fumonisins

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 also produced by Fusarium species, patulin pro-
II. Ochratoxin A-Producing Fungal Species . . . . 353 duced mainly by Penicillium species and ochra-
III. Structure and Biosynthesis of toxin A produced by Aspergillus and Penicillium
Ochratoxin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 species. Especially ochratoxin A attracted much
IV. Physiological Conditions for Production
and Occurrence of Ochratoxin A . . . . . . . . . . . . 357 attention in recent time which led to a consider-
V. Molecular Biology of Ochratoxin A able progress in the knowledge about this sec-
Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 ondary metabolite. Ochratoxin is mainly a
VI. Regulation of Expression of Ochratoxin nephrotoxic mycotoxin but has also hepatotoxic,
A Biosynthesis Genes in Penicillium immunotoxic and teratogenic activities (Pfohl-
in Relation to Environmental Factors . . . . . . . 363
VII. Regulation of Ochratoxin A Biosynthesis Leszkowicz and Manderville 2007). Ochratoxin
in Penicillium in Relation to Light . . . . . . . . . . 368 A is considered to be a human carcinogen of
VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 the 2B class (Petzinger and Weidenbach 2002).
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 It is discussed that ochratoxin A is implicated in
an endemic disease in certain areas of the Bal-
kan, the so-called Balkan endemic nephropathy.
I. Introduction The symptoms which develop during that disease
very much resemble the symptoms of ochratoxin
A toxicity (Pfohl-Leszkowicz et al. 2002). For that
Ochratoxin A was first described as a toxic me-
reason statutory limits were recently set by the
tabolite produced by Aspergillus ochraceus, giv-
European Union. The biological background of
ing this secondary metabolite its name (van der
aflatoxin, trichothecene and fumonisin biosyn-
Merwe et al. 1968). Secondary metabolites of
thesis is well elucidated; however despite of the
filamentous fungi can be more or less artificially
importance of ochratoxin A, surprisingly not that
divided in antibiotics (toxic for microorgan-
much is known about this secondary metabolite.
isms), phytotoxins (toxic for plants) and myco-
This chapter tries to summarize the current
toxins (toxic for humans and animals). About
knowledge about the biology of ochratoxin bio-
300–400 secondary metabolites, which are cate-
synthesis.
gorized as mycotoxins, are known; however most
of them do not have an impact on the health of
human and animal beings, simply because they
do not occur in substantial amounts in foods and II. Ochratoxin A-Producing Fungal
feeds. The most important mycotoxins from that
respect are the aflatoxins, which were the first Species
mycotoxins discovered, because of the death of
100 000 turkeys in Great Britain, described Ochratoxin A was first discovered from a culture
by Asao et al. (1963). Other important mycotox- of A. ochraceus (van der Merwe et al. 1965). These
ins are trichothecenes produced by several authors analyzed the toxicity of five strains of A.
ochraceus. This species is a saprophyte which
1
often occurs on decaying plant material. However
Max Rubner Institute, Federal Research Institute of Nutrition
and Food, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany;
A. ochraceus can also grow on stored wheat, as
e-mail: Rolf.Geisen@MRI.Bund.de, Markus.Schmidt-Heydt@MRI. long as the moisture content is above 16%. It was
Bund.de also reported as a contaminant of “katsuobushi” a

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
354 Rolf Geisen and Markus Schmidt-Heydt
traditional Asian fermented fish. Of the analyzed
five strains, three caused the death of test animals
after feeding; and an LD50 of 0.5 mg/g was ob-
served. This work described the formula of ochra-
toxin for the first time. A. ochraceus has long been
regarded as a major ochratoxin A-producing or-
ganism on plant commodities like coffee beans,
cocoa, grapes and spices. This species belongs to
Aspergillus, Section Circumdati. However, after
detailed examination of the taxonomic relation-
ships, new ochratoxin A-producing species within
this section were recently described (Frisvad et al.
2004). According to these authors eight species of
this section consistently produce large amounts of
ochratoxin A, in particular A. cretensis, A. floccu-
losus, A. pseudoelegans, A. roseoglobulosus,
A. westerdijkiae, A. steynii, A sulphorosus and
Neopetromyces muricatus. Two other species pro-
duce moderate amounts of ochratoxin A, but
less consistently, e.g. A. ochraceus, the original
ochratoxin-producing species, and A. sclero-
tiorum. Four other species produce only very
inconsistently ochratoxin A to a very low amount,
in particular A. melleus, A. ostianus, A. petrakii
and A. persii. In the Aspergillus Nigri section two
very important ochratoxin-producing species are
well known: (i) A. carbonarius is a strong
and consistent ochratoxin A producer and (ii)
A. niger produces inconsistently lower amounts
of ochratoxin. A. niger is a GRAS organism (gen-
erally recognized as safe) which is used extensive-
ly for biotechnological processes like the
production of citric acid, hydrolytic enzymes like
pectinases, proteases and amylases. However in
1994 it was shown for the first time that this
species is also able to produce ochratoxin A
(Abarca et al. 1994). These authors analyzed 19
strains of A. niger isolated from feed and identi-
fied two strains which were able to produce ochra-
toxin A. Both strains produced low amounts of
ochratoxin A, in particular 0.21 mg/g and 0.36 mg/
g. Generally only about 6% of the naturally
isolated strains of A. niger are able to produce
ochratoxin A (Schuster et al. 2002). These facts
must be taken into account when strains of A.
niger are selected for biotechnological purposes.
However, because of the low incidence of ochra-
toxinogenic strains within this species, it should
not be a problem to select suitable technological
strains. The situation with A. carbonarius is
completely different. According to many reports
in the literature >90% (up to 97%) of the
A. carbonarius strains isolated from the natural
environment are able to produce ochratoxin A
(Abarca et al. 2003), which is therefore a very
consistent feature in this species. Recently Sam-
son et al. (2004) updated the taxonomic situation
of the ochratoxin A-producing species within
Section Nigri and described new ochratoxigenic
species, two of them (A. lacticoffeatus, A. sclero-
tioniger) produces high amounts of ochratoxin
A. According to these authors, four of the 15
accepted species within this section are ochra-
toxin producers, namely A. carbonarius, A. niger,
A. lacticoffeatus and A. sclerotioniger. The taxon-
omy with section Nigri is not yet completely
solved (Abarca et al. 2004). For this reason occa-
sionally members of this group, like A. japonicus
and A. foetidus (which are usually not regarded as
ochratoxigenic species), have been reported
to produce ochratoxin A. Recently two new mem-
bers of this section were described, A. uvarum
(Perrone et al. 2008) and A. ibericus (Serra et al.
2006), but neither produces ochratoxin A. Accensi
et al. (1999) described an RFLP of the ITS 1 region
which can distinguish between potential
ochratoxin A-producing A. niger strains (N-type
pattern) and the highly related, but non-ochra-
toxigenic A. tubingensis strains (T-type pattern).
Other described ochratoxin A-producing Asper-
gillus species are A. alliaceus, A. sulphurus,
A. albertensis, A. auricomus and A. wendtii
(Varga et al. 1996).
In contrast only two species of Penicillium
which produce ochratoxin A are known: P.
verrucosum and P. nordicum. Before the clear
clarification of the taxonomic status of ochra-
toxin A-producing Penicillium species by Pitt
(1987), ochratoxin A-producing Penicillia were
assigned to the species P. viridicatum. Ciegler
et al. (1973) first described three subgroups with-
in P. viridicatum, based on the production of
ochratoxin A and other metabolites; two of
these subgroups which were able to produce
ochratoxin A now form P. verrucosum (Pitt
1987) or P. nordicum. P. verrucosum has long
been regarded as the sole Penicillium species
able to produce ochratoxin A. However it was
recently demonstrated by chemotaxonomical
and molecular methods that P. nordicum is clear-
ly a separate species with characteristics distinct
from P. verrucosum (Larsen et al. 2001; Castella
et al. 2002). P. verrucosum and P. nordicum are
morphologically very similar. That is the reason
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis
why most P. nordicum strains were formerly
identified as P. verrucosum by morphological
means. However nowadays rapid and objective
molecular diagnostic methods exist to differenti-
ate between the two species (Bogs et al. 2006).
Also both species exhibit a different chemotype
after analysis of the produced secondary meta-
bolites. P. nordicum for example is able to pro-
duce anacines, sclerotigenin and ochratoxin A,
whereas P. verrucosum synthesizes ochratoxin A,
verrucines and citrinin. In addition both species
differ considerably in their genome organization,
because they showed consistent clearly distin-
guishable RAPD and AFLP profiles (Castella
et al. 2002). Generally most strains of P. nordi-
cum produce high amounts of ochratoxin, where-
as the incidence of ochratoxin A production in P.
verrucosum is much less pronounced. From a
natural population of P. verrucosum only 66%
were able to produce ochratoxin A (Frisvad
et al. 2005). Moreover most strains produce
only moderate amounts of the toxin. However,
because of their frequent occurrence in certain
plant commodities, P. verrucosum plays an im-
portant role as an ochratoxin A-producing con-
taminant.
The Aspergilli and Penicillia able to produce
ochratoxin A are generally not plant pathogens,
but rather saprophytes or maximally opportunis-
tic pathogens which can colonize wounds or
plant material already disturbed by more serious
pathogens or insects. This is especially true for
the Aspergilli, which can for example colonize
bunches of grapes and produce ochratoxin A in
that environment (Battilani et al. 2003; Varga and
Kozakiewicz 2006). The ochratoxigenic Penicillia
in contrast are not regarded as pathogens, but as
so-called storage fungi, colonizing the product
only after harvest. That is especially the case for
cereals. This plant product is frequently colo-
nized by P. verrucosum. In fact P. verrucosum is
the only confirmed ochratoxin A-producing spe-
cies in this habitat. Occasionally found Aspergilli
do not contribute to ochratoxin A biosynthesis in
this environment (Frisvad et al. 2005). Generally
the geographical habitats differ between ochra-
toxin A-producing Aspergilli and Penicillia.
Aspergilli are more adapted to warmer tempera-
tures. Some of them have their growth optimum
between 30 C and 37 C. For this reason they
mainly occur on plants growing in regions with
tropical and subtropical climate like coffee,
355
Table 17.1. Occurrence of ochratoxin A-producing fungi
Habitat/product Species
Cereals Penicillium verrucosum
Salt-rich, dry-cured P. nordicum
meat, cheese
Grapes, wine Aspergillus niger, A. carbonarius,
A. ochraceus
Coffee, spices A. ochraceus, A. carbonarius,
A. niger, A. westerdijkiae,
A. steynii
grapes spices or cocoa. Penicillia grow better at
moderate temperatures, e.g. between 20 C and
25 C. Therefore they can be isolated mainly
from sources of the northern hemisphere. Espe-
cial problems with this fungus have the north
European countries like Denmark, Sweden or
the United Kingdom (Frisvad 2005). Until now
only a few isolates have been found in locations
with higher temperature, like Italy, Spain, Portu-
gal, Nicaragua and Bulgaria (Frisvad et al. 2005).
P. nordicum is a special case because it rarely
occurs on plant material. It is rather adapted to
man made environment. That is the production
plants of salt-rich, dry-cured foods like cheeses
and meats. It is a moderate xerophilic species and
likes a certain concentration of salt. It even shows
increasing growth rates at concentrations of
more than 40g/L NaCl per liter growth medium.
Beside the temperature preferences also the
substrate directs the species which can grow.
Table 17.1 shows the typical occurrence of ochra-
toxin A-producing Aspergilli and Penicillia.
III. Structure and Biosynthesis
of Ochratoxin A
Ochratoxin is a composite mycotoxin consisting
of a polyketide part and the amino acid phenylal-
anine. Chemically ochratoxin A is the pentaketide
3,4-dihydro-3-R-methyl-isocoumarin linked via
its 12-carboxyl group by an peptide bond to the
amino acid phenylalanine (Steyn 1993) The
full nomenclatural name is L-phenylalanine-
N-[5-chloro-3,4-dihydro-8-hydroxy-3-methyl-
1-oxo-1H2-benzopyran-7-yl]carbonyl-R-isocou-
marin (Chemical Abstracts, No. 303-47-9).
Ochratoxin A is chlorinated at position 5 of the
polyketide. Various other forms have been identi-
fied, including ochratoxin B, which carries a
356 Rolf Geisen and Markus Schmidt-Heydt
Fig. 17.1. Structural formulas of ochratoxin A, B and C
(from top to bottom)
hydrogen atom instead of chlorine at position 5.
Ochratoxin C is the ethylester of ochratoxin A
(Fig. 17.1). Ochratoxins a and b respectively also
differ in the chlorination at position 5, however in
contrast to ochratoxin A and B, they are not linked
to phenylalanine.
Ochratoxin A is the most abundant end-prod-
uct synthesized by strains isolated from nature,
followed by ochratoxin B. The a and b forms are
obviously precursors during the biosynthesis of
ochratoxin A (Harris and Mantle 2001); however
they are not produced as end-products. Until
today the biosynthesis of ochratoxin A has not
been completely described in detail. Three major
steps must be involved in ochratoxin A biosynthe-
sis: i.e. (i) biosynthesis of the polyketide, (ii) pep-
tide formation between the polyketide and the
amino acid phenylalanine, which is derived from
the shikimic acid pathway and (iii) chlorination to
ochratoxin A. Today the order of this reactions is
still not completely clear. Huff and Hamilton
(1979) proposed a pathway based on feeding stud-
ies with labeled precursors. The dihydroisocou-
marin polyketide moiety is synthesized by a single
multifunctional enzyme, the ochratoxin A polyke-
tide synthase. Acetate or malonate precursors are
added in a stepwise head to tail addition until the
pentaketide is formed. Intermediates of this reac-
tion have not been detected. Huff and Hamilton
(1979) proposed mellein as an end-product of the
polyketide pathway.
Harris and Mantle (2001) identified the transitory pro-
duction of mellein during the biosynthesis of ochratoxin
A in liquid medium, but not during biosynthesis of
ochratoxin A on solid medium. For this reason the
authors were not sure whether mellein plays an impor-
tant role during the biosynthesis of ochratoxin A.
According to the scheme of Huff and Hamilton (1970)
7-carboxymellein is further methylated via S-adenosyl-
methionine at position 7 to synthesize 7-carboxymellein
or ochratoxin b. This reaction might be carried out
through the action of a methyltransferase domain from
the polyketide synthase as it is the case with the related
mycotoxin citrinin (Shimizu et al. 2005). In fact the
polyketide moiety of citrinin is very similar to that of
ochratoxin b. Both polyketides differ only in the pres-
ence (citrinin) or absence (ochratoxin A) of the methyl
groups. As a next step, Huff and Hamilton (1979) sug-
gested a chloroperoxidase reaction for the addition of the
chlorine to position 5 of the isocoumarin ring system.
The chlorine is part of the structure of ochratoxin A and
ochratoxin C. According to Harris and Mantle (2001),
either ochratoxin b (7-carboxymellein) or ochratoxin B
can be chlorinated to give rise to ochratoxin A. These
authors suggest a major pathway from ochratoxin b to
ochratoxin a to ochratoxin A with a possible branch
from b to B. This would explain the frequent occurrence
of ochratoxin B which can be found in fungal cultures.
However ochratoxin B can also be generated from ochra-
toxin A non-specifically. No clear evidence was found if
this conversion is true to a biotransformation process.
(Harris and Mantle 2001). However in this case ochra-
toxin a is not needed for the biosynthesis, but it can be
identified naturally. According to the results of these
authors mellein per se seems not to be involved in the
biosynthesis pathway. Wei et al. (1971) showed that
chlorine-36 is most effectively incorporated into ochra-
toxin A, when added to the cultures at day two or three.
According to this experiment only ochratoxin A is
labelled by the precursor. In a similar experiment Searcy
et al. (1969) showed that radiolabeled phenylalanine-
1-14C was readily incorporated into the end-product
ochratoxin A and also sodium acetate-2-14C. Both are
the expected main precursors of the pathway.
In the scheme of Huff and Hamilton (1979), the
next step is the joining reaction between the
amino acid phenylalanine and the polyketide
dihydroisocoumarin. Huff and Hamilton (1979)
described an ochratoxin A synthetase as the enzy-
matic function performing these step. This ochra-
toxin synthetases is probably a non-ribosomal
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis
peptide synthetase which ligates the amino acid to
the polyketide and forms the amid bond between
the amino group of phenylalanine and the carboxy
group of 7-carboxymellein. However the ethyles-
ter of phenylalanine seems to be involved in this
reaction. The esterification of the carboxy group
of phenylalanine may be important for protection
purposes (Harris and Mantle 2001). This reaction
would result in ochratoxin C in which the carboxy
group is still esterified. In a later step this ester can
be hydrolyzed to form ochratoxin A. Ochratoxin C
has been isolated from A. ochraceus cultures.
Taken the published facts together, the penta-
ketide ochratoxin b and its chlorinated derivative
ochratoxin a are methylated via the polyketide
synthase reaction. Feeding experiments have
shown that ochratoxin a can be converted into
ochratoxin A, but ochratoxin b can be converted
to ochratoxin A and B. That means that the chlor-
inating step is intermediate and prior to the liga-
tion of the polyketide to the amino acid
phenylalanine. This is described for A. ochraceus,
however new molecular evidence obtained in the
case of Penicillium suggests a slightly different
order of reactions, with the chlorination as the
last reaction before excretion. This is described
below (Fig. 17.2).
IV. Physiological Conditions
for Production and Occurrence
of Ochratoxin A
Like all mycotoxins, ochratoxin A is only pro-
duced under certain growth conditions and is
influenced by growth phase, substrate, tempera-
ture, water activity and pH. Moreover the produc-
tion profiles of mycotoxins are species-specific
and may even vary from strain to strain. Despite
these variability, data about the influence of envi-
ronmental parameters on the biosynthesis of
ochratoxin A are important for estimating the
activation of mycotoxin biosynthesis under a
given situation. Because of this aspect, a wealth of
literature data is available describing ochratoxin A
biosynthesis under a given set of conditions.
One of the most important influences is the
growth substrate. Skrinjar and Dimic (1992)
showed that the nutrient rich YES medium was
an adequate medium for the biosynthesis of
ochratoxin A for A. ochraceus, A. sclerotiorum,
357
A. sulphurus and P. verrucosum. More natural
model media (e.g. rice meal, oat meal, corn grits)
supported ochratoxin A biosynthesis to a much
lesser extent. The same could be shown for
P. nordicum. Also with this species YES was the
most supportive medium for ochratoxin A bio-
synthesis (Geisen 2004). Two other media with
strong regulatory influence on ochratoxin A bio-
synthesis gene expression have been developed
(Geisen et al. 2004). Both are minimal media
with the same composition. Only differences in
the carbon and nitrogen sources exist. One medi-
um (MMp) contains NH4+ as nitrogen source and
glycerol as carbon source and is fully supportive
for ochratoxin A biosynthesis, whereas the other
medium (MMr) contains the combination NO3–/
glucose and nearly completely represses the
toxin biosynthesis, albeit growth is still possible.
Expression analysis of ochratoxin A biosynthesis
genes revealed that, after growth in the supportive
medium with glycerol and NH4+, ochratoxin
transcript levels are several-fold higher compared
to the non-supportive medium (Geisen et al. 2006).
These results indicate that glucose represses and
NH4+ activates ochratoxin A biosynthesis in Peni-
cillium. The effect of glucose on the expression of
secondary metabolite genes is well known. For
example the biosynthesis of the antibiotic actino-
mycin is tightly regulated by catabolite repression
(Gallo and Katz 1972; Yu and Keller 2005). Catabo-
lite repression is mediated via the creA zinc finger
DNA binding factor (Dowzer and Kelly 1991)
which binds to a consensus sequence located in
front of regulated promoters, thereby down-regu-
lating transcription of the respective gene. Accord-
ing to the results described above, this seems to be
the case for the biosynthesis of ochratoxin A. In
media containing glucose as sole carbon source,
ochratoxin A biosynthesis is strongly inhibited.
Binding sites for the CREA protein have been de-
scribed in Penicillium (Diez et al. 2005).
In contrast to the situation of ochratoxin A, the biosynthe-
sis of aflatoxin seems not to be regulated by carbon catab-
olite repression. According to Bhatnagar et al. (2006)
aflatoxin biosynthesis by A. flavus and A. parasiticus is
activated by simple carbon sources like glucose and su-
crose, but not by peptone or complex carbohydrates like
starch. Interestingly in case of aflatoxin a hexose utiliza-
tion gene cluster is located just adjacent to the aflatoxin
biosynthesis gene cluster, indicating a possible relation-
ship. Generally the regulation of aflatoxin biosynthesis by
simple carbon sources is not well understood. In contrast
358 Rolf Geisen and Markus Schmidt-Heydt

O
O O OH O

CH3 C S CoA C C
HC C S Enzyme O
Polykctide synthasc 2
H
2
O O O H
C C CH
O CH3
4X CO C C CH3
CH2 C S CoA 2
H2 H2

CH3

OH O OH O OH O

H3C COOH
O O O

H H H
CH3 CH3 CH3

OH O OH O O O CH O

COOH COOH O P C
O Chloroperoxidase O O
O

CH3 CH CH3
3

CI CI

COOH O
H H H2
2 2
C CH C C O C CH3

Shikimic Acid Pathway NH2 NH2

Phenylalanine

O O O CH O
H2 H2
C C O C CH3 O P C
O
Peptide synthetase O
NH2
H
CH3

CI

O
H
2
HC O C CH3 O CH O
H
2
C C N C
O
H H
H
CH3
Ochratoxin C
CI
O
H2
HC O C CH3 O CH O
H
2
C C N C
O
H H
H
CH3

CI

H
2
3HC C OH

COOH O CH O
H2
C C N C
O
H H
H
CH3
Ochratoxin A

Fig. 17.2. Proposed biosynthesis pathway according to the literature data mentioned above. The molecular data of
Penicillium imply that the chlorine atom is incorporated during the very last reaction, shortly before the excretion of the
molecule into the environment

to the regulation of many other secondary metabolites, promoters, which explains the independence from carbon
aflatoxin biosynthesis is activated by these simple carbo- catabolite repression. However CREA may play a role in
hydrates. Most of the aflatoxin biosynthesis genes studied the regulation of the expression of an antisense transcript
so far do not have CREA binding sites in front of their of the regulatory factor aflR (Bhatnagar et al. 2003).
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis
Ehrlich et al. (2002) reported a few putative CREA sites
identified in the intergenic region between the nor-1–
pksA genes of A. nomius, A. bombycis, A. pseudotamarii
and A. flavus strain SBG. These are all less important and
“atypical” aflatoxin-producing species. According to
present evidence, CREA regulation seems not to play an
important role during aflatoxin biosynthesis, which is
in contrast to the results found for ochratoxin A biosyn-
thesis in Penicillium, in which CREA seems to play a role.
Tudzynski et al. (2000) characterized the creA genes
from two plant-pathogenic fungi, in particular from Gib-
berella fujikuroi and Botrytis cinerea, the former produc-
ing the secondary metabolite gibberellin (Tudzynski et al.
2000).
Also the nitrogen source plays a prominent role in
regulating mycotoxin biosynthesis. In the case of
Penicillium, NH4+ increases and NO3– decreases
the biosynthesis of ochratoxin A. This is also the
case for aflatoxin biosynthesis. Ehrlich and Cotty
(2002) described that a strain of A. flavus SBG
produced 20-fold lower amounts of aflatoxin in
buffered nitrate medium than in ammonium salts
medium. This effect was not true for a changed
expression of the regulatory gene aflR, but the
expression of the coregulatory gene aflJ was re-
duced by a factor of two. The promoter region of
the aflJ genes carries some potential AREA bind-
ing sites. AREA is a global regulatory protein
which can regulate expression of certain genes in
response to the presence (repressed) or absence
(induced) of NH4+ (Marzluf 1997).
According to Price et al. (2005), organic nitrogen sources
induce, while inorganic sources retard aflatoxin biosyn-
thesis. According to these authors, nitrate repression may
occur due to an altered redox state in the cell. By micro-
array analysis during aflatoxin production conditions, a
potential regulon was identified, which was upregulated
under growth on nitrate medium. One gene of this regulon
may be involved in heme biosynthesis. Heme is a structur-
al component of nitrate reductase, which is the first en-
zyme in the catabolic pathway leading from nitrate to
ammonia. This observation might constitute a link to the
nitrate repression of many mycotoxin biosynthesis path-
ways. Other genes of the regulon code for proteins
involved in the electron transport system, which might
play a role during reduction of nitrate to ammonia. Also,
the genes in this regulon carry AREA binding sites in their
promoters, indicating their coregulation. Bhatnagar et al.
(2006) also described AREA binding sites in the promoter
elements of aflJ and aflR. The recognition site has the
GATA consensus motive. Certain strains of A. flavus
which respond differentially to nitrate had differences in
the number of GATA sites within the promoter region of
aflJ. According to Bhatnagar et al. (2006), the direct cause
of nitrogen regulation is still not clear, but it could be
mediated by AREA.
359
Orvehed et al. (1988) described the inhibiting
effect of NaNO3 on alternariol biosynthesis by
Alternaria alternata. The biosynthesis of the
secondary metabolite gibberellin by G. fuji-
kuroi is strongly suppressed by high amounts
of nitrogen (e.g. ammonium, glutamate, gluta-
mine, nitrate). For this fungus it has been
shown that AREA controls expression of the
gibberellin biosynthesis genes. The expression
of the gibberellin biosynthesis genes in mutants
of the areA-Gf gene is strongly reduced, indi-
cating its involvement in regulation (Tudzynski
2005).
These examples pinpoint the importance of
AREA regulation on the transcription of second-
ary metabolites including mycotoxins. Recently
an areA/nmrC homologue was identified in P.
nordicum, which is upregulated under NO3– and
downregulated under NH4+ growth condition.
Therefore all experimental evidence suggest that
a similar mechanism is responsible for nitrate
suppression of ochratoxin A biosynthesis in
these fungi.
Another external factor which plays an impor-
tant role on ochratoxin A biosynthesis is the pH.
Arroyo et al. (2003) reported that the biosynthesis
of ochratoxin A increases in A. ochraceus when
the pH drops to a range of 6.0–4.5. O’Callaghan
et al. (2006) also found at the phenotypical and
molecular level that biosynthesis gene expression
and ochratoxin A biosynthesis was higher at low
pH values (down to pH 4.0) than at neutral or
slightly alkaline conditions. This is in contrast to
the situation in Penicillium. P. nordicum shows
highest production at neutral to slightly alkaline
conditions, e.g. between pH 6.0 and pH 8.0 (Geisen
2004), whereas production is reduced under acidic
conditions (<pH 5.0). In case of aflatoxin biosyn-
thesis also acidic conditions favor toxin biosyn-
thesis (Bhatnagar et al. 2006). Aflatoxin
biosynthesis by A. flavus is highest at a pH range
between 3.4 and 5.5. Keller et al. (1997) found a
five- to tenfold increase of aflatoxin biosynthesis
in A. parasiticus and sterigmatocystin synthesis
in A. nidulans, when cultures were grown at
pH 4.0–5.0 versus pH 8.0. These authors also ana-
lyzed the expression of certain key genes of the
biosynthesis pathways of these secondary meta-
bolites and showed that stcU (a gene of the ster-
igmatocystin pathway) and ver-1 (a gene of the
aflatoxin pathway) were upregulated under acidic
conditions.
360 Rolf Geisen and Markus Schmidt-Heydt
Interestingly the influence of pH on the biosynthesis of
ochratoxin A is clearly detectable but much less pro-
nounced than the influence of temperature and water
activity. This might be true because of the presence of a
pH controlled system which in turn regulates downstream
genes, e.g. the pacC/palA, B, C, F, H, I system as elucidated
in A. nidulans. The pacC gene codes for a transcription
factor, which acts downstream of the six pal genes. The
products of these genes are components of a signal trans-
duction pathway. PALI and PALH are potential plasma
membrane pH sensors. PACC can either activate or re-
press downstream genes (Arst and Penalva 2003). In a
microarray expression analysis of genes upregulated
under aflatoxin-producing conditions in A. flavus, Price
et al. (2005) found genes with creA, areA and pacC tran-
scription factor binding sites in their promoter regions,
indicating the involvement of this mechanism in the bio-
synthesis of the mycotoxin aflatoxin.
Ehrlich et al. (2002) analyzed the expression of
pksA an acid-expressed gene, involved in the
biosynthesis of aflatoxin in A. flavus. Regulation
of acid- or alkaline-regulated genes is a process
involving the proteolytic degradation of the
PACC protein, giving rise to the active form
that supports the expression of alkaline-activated
genes and represses the expression of acid-acti-
vated genes. Since the biosynthesis of aflatoxin is
high under acidic conditions, but repressed
under neutral and alkaline conditions, this mech-
anism seems to act negatively on genes of the
pathway containing PACC binding sites. This
was confirmed by the fact that a deletion of one
or more of the PACC binding sites in the pro-
motor region of pksA mimics acid conditions.
Albeit no molecular evidence is available yet for
ochratoxin A biosynthesis in Penicillia, just the
opposite seems to be true for that fungal species.
The ochratoxin A biosynthesis genes seems to
be regulated positively by PACC under alkaline
conditions, indicating that these genes are
alkaline-activated. Fusarium oxysporum, another
mycotoxin-producing fungus, also contains a
characterized homologue of the pacC gene
(Caracuel et al. 2003) which is involved in
virulence regulation.
V. Molecular Biology of Ochratoxin
A Biosynthesis
Despite the importance of ochratoxin A as a my-
cotoxin for human health, not much is known
about the genetical background of ochratoxin A
biosynthesis, especially in Aspergillus, compared
to the other important mycotoxins like aflatoxin,
trichothecenes or the fumonisins. In contrast the
situation in Penicillium is somewhat further elu-
cidated.
Generally the genes for the biosynthesis of
mycotoxins are organized in clusters (Proctor
et al. 2003; Yu et al. 2004; Brown et al. 2005).
One or two regulatory genes (regulated by global
regulatory factors as mentioned above) control
the expression of the mycotoxin biosynthesis
structural genes and activate these genes under
permissive conditions (Fig. 17.3).
Recently a polyketide synthase responsible for
the biosynthesis of ochratoxin A in A. ochraceus
was partly characterized (O’Callaghan et al. 2003).
These authors identified a DNA fragment of 1.4 kb
which shared 28–35% identity to acyl transferase
regions from fungal polyketide synthases. Expres-
sion analysis of this gene revealed that it is only
activated under conditions which are supportive
for ochratoxin A biosynthesis by A. ochraceus.
The expression could also only be found at the
early phases of ochratoxin A biosynthesis. More-
over a transformant in which the pks gene was
inactivated by integration was no longer able to
produce ochratoxin A, giving evidence for the
involvement of this gene in ochratoxin A biosyn-
thesis. The authors used an SSH-PCR approach
(suppressive substractive hybridization PCR) to
identify the pks gene, by using permissive cDNA
as tester and restrictive cDNA as driver. Before
identifying the correct pks gene, the authors
cloned three other pks genes which were actively
transcribed, but were not involved in ochratoxin
biosynthesis. This indicates that A. ochraceus con-
tains several polyketide synthases, as was also
found in other fungi like for example G. zeae
(Gaffor et al. 2005). The work by Atoui et al.
(2006) clearly shows that the two ochratoxin A-
producing species A. ochraceus and A. carbonarius
carries several pks genes. These authors used sev-
eral primer sequences specific for ketosynthase
(KS) domains from pks genes and could identify
nine different KS domain sequences in A. ochraceus
and five in A. carbonarius. In a phylogenetic tree
based on the similarity of the DNA sequences of
the domains, the identified gene fragments were
distributed over five different similarity clusters,
indicating different functions of these enzymes.
No hint for the involvement of any of these
genes in ochratoxin A biosynthesis was given in
this report. Based on one of these pks genes, a
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis
Specific
regulatory gene
Global transcription factors
responsive against changes in
water activity
pH
light
substrate
Fig. 17.3. Organization of a typical secondary metabolite gene cluster. The arrows indicate the hierarchical regulation of
global versus specific transcription factors. Global factors are responsive to environmental changes
molecular detection system to specifically identify
A. carbonarius on infected grapes was described
(Atoui et al. 2007). Beside the polyketide synthase
of A. ochraceus, whose expression is correlated
with the production of ochratoxin A (O’Callaghan
et al. 2003), the same authors identified two p450
monooxygenase genes, in particular p450-H11
and p450-B03 which had a very similar expression
profile compared to the polyketide synthase gene
(O’Callaghan et al. 2006). For this reason the
authors conclude that both gene products might
be involved in ochratoxin A biosynthesis. These
three genes have been identified using an SSH
approach, with which 152 genes, upregulated
under ochratoxin A-producing conditions have
been found. In a later analysis the same authors
compared the sequences of the ochratoxin A pks
gene of A. ochraceus, with that of other known pks
genes (O’Callaghan 2006b). They found that the
ochratoxin A pks gene has a high homology, espe-
cially in the ketosynthase domain, with pks genes
of the MSAS type (methyl salicylic acid synthase).
That is in nice agreement with the otapksPN gene
identified in P. nordicum (see below), which also
shows high homology to MSAS type pks genes
(Karolewiez and Geisen 2005).
Another report of the cloning of an adjacent
fragment of the same gene was given by Dao et al.
(2005). They used the identified DNA fragment for
the development of primers specific for ochra-
toxin A-producing fungi. These primers were
used in diagnostic PCR reactions to identify po-
tential ochratoxin A-producing organisms in en-
vironmental samples.
In the case of P. nordicum the gene cluster
responsible for ochratoxin A biosynthesis is partly
elucidated (Karolewiez and Geisen 2005; Geisen
361
Gene cluster
et al. 2006). Figure 17.4 shows the information
currently available about the gene cluster in
P. nordicum.
Interestingly the presence of homologues of
the genes has also been demonstrated for P.
verrucosum, the closely related species. All the
genes seem to be very similar, except the polyke-
tide synthase which differs considerably between
the two species. Part of a polyketide synthase from
P. verrucosum CBS 302.48, obviously involved in
ochratoxin A biosynthesis, was deposited in Gen-
eBank under accession number EF527420. This
fragment covers the acyltransferase domain of
the polyketide synthase of P. verrucosum and has
only limited homology to the respective domain of
P. nordicum (similarity index 13.7, after the Mar-
tinez/Needleman_Wunsch DNA alignment). If
this dissimilarity between the two polyketide
synthases of the two related species is due to the
fact that P. verrucosum is also able to produce
citrinin, another polyketide mycotoxin is not yet
clear. It is speculated that the P. verrucosum poly-
ketide synthase might be involved in the biosyn-
thesis of both toxins. The identified genes of
P. nordicum involved in ochratoxin A biosynthe-
sis are a polyketide synthase (otapksPN), a non-
ribosomal peptide synthetase (otanpsPN), a gene
with some homology to chlorinating enzymes
(otachlPN; which is thought to be involved in the
chlorination step) and a gene with high homology
to the organic anion transporter (oat) from rat
kidneys (otatraPN). The gene product of the rat
gene is described to be involved in the export of
ochratoxin A from kidney cells. This gives a
strong indication that the P. nordicum gene
might be involved in the transport of ochratoxin
A out of the fungal cell. Until now no gene or gene
362 Rolf Geisen and Markus Schmidt-Heydt
P. nordicum
otanpsPN otapksPN
P. verrucosum
otanpsPV otapksPV
P. nalgiovense
otanpsPNa otapksPNa
Fig. 17.4. Schematic drawing of the ochratoxin A gene clusters in P. nordicum, P. verrucosum and P. nalgiovense. For the
dotted line no sequence information is available yet
product involved in the regulation of ochratoxin A
has been described. The regulation of activation of
the identified genes very congruently fits to the
phenotypic production of ochratoxin A; and gene
inactivation approaches demonstrate the involve-
ment of three of the identified genes in ochratoxin
A biosynthesis. Transformation of P. nordicum
with either internal fragments of the otapksPN,
the otanpsPN or the otachlPN genes revealed
strains unable to produce ochratoxin A, in con-
trast to the wild type. Interestingly, in the case of
inactivation of the otachlPN gene, the respective
transformant produced mainly ochratoxin B in-
stead of ochratoxin A. This indicates the possible
involvement of the gene product in the chlorina-
tion step.
Very unexpectedly homologues of these genes
have not yet been identified in the major ochra-
toxin A-producing Aspergilli, indicating a conver-
gent evolution of the non-homologous gene
clusters, rather than separation from a common
ancestor. Convergent evolution of secondary me-
tabolite pathways is also suggested by Frisvad
et al. (2008), trying to explain the presence of
similar secondary metabolites in phylogenetically
unrelated species. The P. nordicum otapksPN is a
polyketide synthase of the MSAS type (methyl
salicylic acid synthetases), leading to partially re-
duced polyketides, which fits very well with the
structure of ochratoxin A. Atoui et al. (2006)
found a pks gene in A. carbonarius, whose acyl-
transferase domain has about 47% identity to the
PKS responsible for the biosynthesis of methyl
salicylic acid (MSA) by A. terreus and P. patulum
high amounts of OTA
otachlPN otatraPN
moderate amounts of OTA, citrinin
otachlPV otatraPV
no OTA detectable
otachlPNa otatraPNa
and 44% identity to the otapksPN gene from
P. nordicum. However until now it has not
been ultimately shown that this pks gene from
A. carbonarius is involved in ochratoxin A biosyn-
thesis. The original report about the identification
of the otapksPN gene also described highest
homology to the MSAS-like pks genes from A.
terreus, Byssochlamys nivea and P. griseofulvum
(Karolewiez and Geisen 2005). As can be seen
from Fig. 17.4, another species could be found
which obviously carries homologues of the ochra-
toxin A biosynthesis genes. This species is P. nal-
giovense, which is known as a biotechnological
production culture for dry-cured, salt-rich foods,
especially meats. This is the same habitat in which
P. nordicum nearly exclusively occurs. Until now,
P. nalgiovense has never been described to pro-
duce ochratoxin A. Comparison of part of the pks
gene homologue in P. nalgiovense revealed high
homology with P. nordicum. Only two nucleotide
exchanges were found over a stretch of about 460
nucleotides for each of the two analyzed P. nalgio-
vense sequences (Bogs et al. 2006). However a
subsequent expression analysis revealed silent
otapksPNa genes (at least under the conditions
applied) in the two analyzed P. nalgiovense
strains, compared to a very active gene in case of
P. nordicum.
This looks like a situation similar to the A. flavus/A.
oryzea or A. parasiticus/A. sojae species pairs. In these
cases the two former species are potent aflatoxin produ-
cers, whereas the two latter species are regarded as domes-
ticated forms of the two former ones. Interestingly the
latter species are used for food fermentations in Asian
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis
countries and have never been reported to produce afla-
toxin. The reason for its non-aflatoxigenicity has been well
elucidated in the case of A. sojae (Chang et al. 2007). In
this species the interaction between the two regulatory
proteins AFLR and AFLJ is disturbed, leading to a non-
aflatoxigenic phenotype. This effect is caused by an early
termination of the translation of the AFLR protein. In this
case an AFLR variant is formed with a reduced ability for
transcription activation. In addition this variant form is
no longer able to interact with AFLJ. Other mutations are
responsible for the inactivation of aflatoxin biosynthesis
in A. sojae. For example the pksA gene, a polyketide
synthase involved in aflatoxin biosynthesis, is defective
because it contains a pretermination stop codon which
leads to a truncated product of 1847 amino acids. In the
case of A. oryzea this effect has not been shown yet.
However Tominaga et al. (2006) recently compared the
aflatoxin gene cluster of A. flavus with that of A. oryzea.
Generally they found a high homology of 97–99% between
both clusters. However three genes showed more pro-
nounced differences. The aflT gene of A. oryzea had a
deletion of 257 bp and there was a frame shift mutation in
nor-A and a base pair substitution in verA. In addition two
substitutions in the promotor region of the aflR gene could
be identified: one in an AREA binding site and one in a
FACB binding site. Obviously these mutations are respon-
sible for the aflatoxin negative phenotype of A. oryzea.
Until now nothing has been known about the
reason for the non-ochratoxin A phenotype of
P. nalgiovense. Similar mutations could be re-
sponsible for this feature, but it is not absolutely
ruled out that P. nalgiovense may be able to pro-
duce ochratoxin under certain growth conditions.
Recently a systematic analysis of the occur-
rence of P. nordicum on salt-rich, dry-cured pro-
ducts isolated a set of strains which were partly
able to produce low amounts of ochratoxin A and
which carried homologues of the ochratoxin A
biosynthesis genes of P. nordicum. Also the pks
genes were similar to those of P. nordicum. How-
ever the morphology and RAPD types differed
from those of P. nordicum, P. nalgiovense and
P. verrucosum. In comparison, P. nordicum pro-
duced high amounts of ochratoxin, whereas this
group of strains produced very low amounts
of ochratoxin A, if at all. These preliminary results
indicate that possibly a third species (as yet unde-
scribed) occurs in this limited habitat of salt-
fermented foods. That is a very interesting fact
which means that two (plus one unidentified) of
four species now known to carry the ochratoxin
A biosynthesis genes occur in this limited habitat
of salt-rich, dry-cured foods. This points to
the intriguing idea that ochratoxin A might be
involved in salt stress adaptation by Penicillia.
363
VI. Regulation of Expression
of Ochratoxin A Biosynthesis
Genes in Penicillium in Relation
to Environmental Factors
Ochratoxin A is produced in the secondary me-
tabolism of the fungus. The distinction between
primary and secondary metabolism is more or
less historical; however from the viewpoint of
expression kinetics secondary metabolism is acti-
vated at later growth phases. Historically trans-
mission of the culture from the actively growing
trophophase state to the more static stationary
phase is regarded as a signal for induction of
secondary metabolite biosynthesis. However re-
cent detailed analysis showed that the production
of mycotoxins, although it does not take place
from the very beginning of growth, can start very
early and at any case before reaching the idio-
phase. This means that signals other than the
shift from tropho- to idiophase must trigger acti-
vation of mycotoxin biosynthesis. The exhaustion
of a nutrient, very often nitrogen, is regarded as
one signal for secondary metabolite activation
(Bennett and Christensen 1983). Shortage of a
nutrient can be regarded as some kind of nutri-
tional stress caused by unfavorable nutrient com-
position of the substrate. This may be occur quite
fast after growth on solid agar medium, when the
diffusion of nutrients plays a role. Likewise on
natural substrates the overall nutrient amount is
high, but nevertheless nutrient limitations in the
microenvironment may also occur due to diffu-
sion problems.
As shown above, several external parameters
like substrate, temperature, water activity, pH,
light, preservatives and fungicides do have an
important influence on the biosynthesis of myco-
toxins. Mycotoxin biosynthesis genes are not con-
stitutively expressed, but are induced under
favorable conditions and growth phases. This
situation implies that these external effects have
an influence on gene transcription. This was
demonstrated for ochratoxin A biosynthesis by
O’Callaghan et al. (2006), who showed that the
composition of the growth medium has a strong
effect on the expression of the ochratoxin A pks
gene of A. ochraceus. After growth on potato
dextrose medium, and on potato dextrose medi-
um supplemented with casamino acids, they
observed a very low expression of the pks gene
364 Rolf Geisen and Markus Schmidt-Heydt
and two additional cytochrome monooxygenase
genes, thought to be involved in ochratoxin A
biosynthesis. In contrast, after growth on yeast
extract medium, expression of these genes were
39-fold higher. The same authors showed that the
effect of external pH on ochratoxin A biosynthe-
sis is also exerted at the transcriptional level. At
low pH values (pH 3–4) when the biosynthesis of
ochratoxin A by A. ochraceus was high, the tran-
script levels also were high, albeit only minor
influences on the produced biomass could be
observed. The transcript level of the pks gene
was about twofold higher at low pH values (pH
3) compared to high pH values (pH 10). In con-
trast, the transcript accumulation of the glyceral-
dehyde-3-phosphate dehydrogenase gene was
similar in all cultures, with about the same copy
number at all conditions.
By microarray O’Brian et al. (2007) analyzed the
expression pattern of an aflatoxin-producing strain of A.
flavus. During this approach the authors analyzed 5000
genes, from which 144 where differentially expressed be-
tween the two analyzed temperatures (28 C, 37 C). It was
already known from phenotypical analysis that the pro-
duction optimum for aflatoxin is 28 C, whereas 37 C is
the growth optimum. Very low amounts of aflatoxin are
produced at the growth optimum. From the 144 differen-
tially expressed genes 103 were more highly expressed at
28 C. About 25% of theses genes were related to second-
ary metabolism. As expected, the authors demonstrated
that all aflatoxin biosynthesis genes are much more
expressed at 28 C compared to 37 C. This decrease in
expression at elevated temperatures explains the very low
to undetectable amounts of aflatoxin produced by A.
flavus at optimal growth conditions of 37 C. However
on other substrates (e.g. ground nuts) the temperature
profile of aflatoxin biosynthesis may be completely differ-
ent. Interestingly some pathway genes (aflR, aflJ, aflS)
showed no differential expression between the two tem-
peratures. For this reason the authors concluded that the
temperature per se does not have an influence on gene
expression, but that less product of the aflR gene is
produced under elevated temperatures. The aflR gene is
the regulatory gene of the aflatoxin biosynthesis pathway.
According to another suggestion by the authors, the
AFLR protein might also be non-functional at higher
temperatures. In another analysis, Price et al. (2005)
used a whole genome microarray to study the influence
of ammonia and pH on aflatoxin gene expression. They
showed that beside the aflatoxin biosynthesis cluster
other regulons are coregulated, indicating accessory in-
volvement in aflatoxin biosynthesis. The authors stated
for example that, for a carbon source to support both
growth and aflatoxin production, it must be accessible to
both the hexose monophosphate and glycolytic pathways.
In the current analysis genes upregulated in response to
sucrose and aflatoxin biosynthesis could be identified.
This situation indicated the sometimes complex mechan-
isms controlling regulation of mycotoxin biosynthesis
genes by environmental factors. This microarray study
could also reveal a possible influence of changes in pH on
aflatoxin biosynthesis. The authors identified a set of
27 genes coordinately up-regulated with increasing pH.
This group of genes also includes pkaA, a phosphokinase
involved in regulation of aflatoxin biosynthesis. The
activity of pkaA inhibits aflatoxin biosynthesis.
For the regulation of ochratoxin A biosynthesis in
Penicillium such an in depth description of the
molecular mechanisms behind the phenotypic
influences cannot be given yet, nevertheless de-
tailed and systematical analysis of ochratoxin A
gene expression have been performed. It can be
shown as expected that the polyketide synthase
gene from P. nordicum is regulated and not con-
stitutively expressed (Geisen et al. 2004). The first
induction of the otapksPN gene can be observed
after 3–4 days of growth on YES medium. The
maximum of expression is reached between day
5 and day 8. After that time-point expression
declines sharply. In contrast, expression of a
house-keeping gene remains constant over the
whole observation period of 10 days. The pheno-
typic production of ochratoxin A by P. nordicum
completely fits to the expression pattern of the
otapksPN gene. Low amounts of ochratoxin A
can be detected by HPLC after 4–5 days of incuba-
tion, i.e. more than 24 h after induction of the gene
took place. As long as the otapksPN gene is
expressed, e.g. until day 8, the concentration of
ochratoxin A increased. After the decline of
otapksPN gene expression, the ochratoxin A con-
centration remains constant over the observation
period (oscillates around a certain level, as dis-
cussed later). The expression behavior of the
otapksPN gene very much resembles the pattern
of the citrinin polyketide synthase of Monascus
purpureus (Shimizu et al. 2005). Also this pks
gene has an expression optimum after 3–6 days
which is followed by citrinin production. The fact
that the expression of both genes is well correlated
with the production of the respective secondary
metabolite demonstrates that both can be
regarded as key enzymes of the pathway. The
transcription activation of mycotoxin biosynthe-
sis genes are not necessarily direct correlated to
toxin biosynthesis. In a detailed analysis, Scherm
et al. (2005) showed that the expression pattern of
only certain genes of the aflatoxin biosynthesis
pathway in A. flavus fits well the phenotypic
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis 365

aflatoxin production, in particular nor-1, omtB

Fig. 17.5. Timely coordinated expression of the putative ochratoxin A biosynthesis genes. The expression kinetics of each gene is shown. The optima are marked by
and omt-1. Also a detailed analysis of the known
ochratoxin A-related genes of P. nordicum

10
revealed different expression kinetics in correla-

9
tion to ochratoxin A biosynthesis (Geisen et al.

8
2006). According to this analysis there seems to be

Time [d]
a timely consecutive occurrence of expression

6
optima in the ochratoxin A-related genes. The

otatraPN
5
4
first gene, whose activation can be detected after
3–4 days and whose optimum is at 5–8 days, is the
3

otapksPN gene, followed by the otanpsPN (activa-

2

tion from day 5 to day 7; optimum days 8–9), the

16000000

14000000

12000000

10000000

8000000

6000000

4000000

2000000

otachlPN (optimum days 8–9) and the otatraPN

gene (optimum day 9; Fig. 17.5).
As mentioned above, the expression pattern of
10

the otapksPN gene fits most to the phenotypical

9

production of ochratoxin A. In addition the

8

expression of the b-actin gene of P. nordicum

7

Time [d]

remains constant over the whole observation pe-

otach/PN
6

riod. It is intriguing to speculate that the consecu-

5

tive occurrence of expression optima of the

4

various genes is a hint for the consecutive enzy-

3

matic reactions during ochratoxin A biosynthesis.

2

According to this order the otapksPN gene would

14000

12000

10000

8000

6000

4000

2000

be activated first, which is logical from the biosyn-

thesis point of view, as its gene product first
10

synthesizes the polyketide moiety dihydroisocou-

9

marin. The second active gene, the otanpsPN

8

gene, after activation adds the amino acid phenyl-

otapksPN
7

Time [d]

alanine, which apparently originates from the shi-

6

kimic acid pathway. The gene product of the

5

otanpsPN gene is a non-ribosomal peptide synthe-

4

tase, whose activity leads to the formation of a

3

peptide bond between phenylalanine and the

polyketide moiety. This reaction gives rise to
2
250000

200000

150000

100000

50000

ochratoxin B. The toxicity of ochratoxin B is by

a factor of 10 to 100 less than the toxicity of
ochratoxin A. At the molecular level ochratoxin
10

A is a competitive inhibitor of the phenylalanine

9

t-RNA synthetase. This might also be the case in

8

otanpsPN

the producer strains. A late chlorination and an

7

Time [d]

immediate excretion might greatly reduce the

6

toxic effect of ochratoxin A on the producer cell.

5

Interestingly the otachlPN and the otatraPN genes

4

have their expression optimum at the latest, which

3

ideally fits with this hypothesis. This putative

order of reactions would include a self-protection
2
14000

12000

10000

8000

6000

4000

2000

mechanism against the toxicity of ochratoxin A.

rectangles

Expression [copy number/mg] The most toxic compounds are formed very late
during biosynthesis and seem to be excreted just
after formation.
366 Rolf Geisen and Markus Schmidt-Heydt
A self-protection mechanism is also known in the biosyn-
thesis of trichothecenes by Fusaria. The toxicity of tri-
chothecenes is also exerted at the level of translation. In
this case the gene tri101 codes for an acetyl transferase,
which acetylates intracellularly the produced trichothe-
cenes and thereby reducing their toxic activities (Kimura
et al. 2007). This gene was originally identified as a clone in
Schizosacharomyces pombe that conferred resistance to T-
2 toxin, a type A trichothecene, when expressed in the
heterologous host. Later however it was shown, that F.
sporotrichioides strains in which this gene was inactivated
were not lethal, but produced another set of trichothecene
end-products (McCormick et al. 1999). For this reason the
authors conclude that the gene product is involved in
additional tasks within the biosynthesis of trichothecenes
besides the self-protection effect. By transgenic expression
of this gene in either tobacco (Muhitch et al. 2000) or
wheat (Okubara et al. 2002), the resistance of these crops
against the toxic action of the trichothecenes has been
increased. A second self-protection mechanism in Fusari-
um is exerted by the tri12 gene product. This protein is an
efflux pump which excretes the toxin out of the cell. This
activation might be analogous to the activity of the ota-
traPN gene.
The influence of external abiotic factors like tem-
perature, water activity and pH on the expression
of the otapksPN gene of P. nordicum (Geisen 2004)
or the otapksPV gene of P. verrucosum have been
extensively studied. Generally after continuously
changing a single parameter the growth rate has a
clear optimum at a certain value and decreases
when the parameter (for example temperature)
approaches suboptimal conditions. This is true
for all parameters analyzed (temperature, pH,
water activity). In contrast to this behavior,
expression of the otapksPV gene has several
peaks of induction (Schmidt-Heydt et al. 2008):
one major peak at or near the optimal growth
conditions and one minor peak under conditions
which impose mild stress to the fungus. This is
indicated by the fact that, under these conditions,
P. verrucosum shows a reduced growth. The phe-
notypic production of ochratoxin A by P. verru-
cosum completely follows this expression
behavior (Fig. 17.6).
This typical expression profile was not only
found in the case of water activity, but also at
changing temperature or pH values. Moreover
the trichothecene-producing Fusarium culmorum
and aflatoxin-producing Aspergillus flavus strains
also exhibited the same profile, demonstrating
that this profile seems to be a general one. Also
after microarray analysis of the whole known
ochratoxin A cluster of genes the same expres-
sion profile was found in relation to changing
parameters, one major peak of expression at
near optimal growth conditions and a smaller
peak at conditions which lead to reduction, but
not to a stop of growth. Obviously the influence of
external abiotic factors, which leads to cellular
stresses activate ochratoxin A biosynthesis. This
microarray expression profile was found for all
three species mentioned above, again indicating
the general importance of this profile. The occur-
rence is interpreted as some kind of stress re-
sponse triggered by environmental parameters
reaching unfavorable conditions for growth. The
major peaks at optimum growth conditions at a
first glance do not look like stress adaptation.
However P. verrucosum produces high amounts
of ochratoxin A on solid agar medium. P. verru-
cosum grows very slowly and in a very compact
form, producing quite high biomass per defined
surface area. This growth morphology leads to the
situation that high amounts of nutrients are need-
ed during the trophophase. On a growth medium
like agar plates, where diffusion plays a role, this
situation might lead to a shortage of nutrients, for
example nitrogen, whose exhaustion is known to
induce mycotoxin biosynthesis (Bennet and
Christensen 1988). It was shown that moderate
growth conditions, e.g. at conditions where
growth is not optimal (but not yet retarded by
unfavorable conditions) produced the lowest
amounts of ochratoxin A. This fact again demon-
strates that certain kinds of stress, either exerted
by abiotic factors or by nutritional constraints
activate ochratoxin A biosynthesis. It is also
known that suboptimal amounts of fungicides
or preservatives, which retard but still allow
growth, lead to an increase in mycotoxin biosyn-
thesis. Ochiai et al. (2007) constructed a Fusari-
um graminearum reporter strain, with a GFP
protein under the control of the tri5 promotor.
TRI5, a trichodiene synthase, is the key enzyme
of the trichothecene biosynthesis pathway. They
used this strain to analyze the influence of vari-
ous fungicides on the activation of trichothecene
biosynthesis genes. According to their results,
triazol fungicides transcriptionally activate the
tri5 gene under sublethal conditions. Similar
results were described by Schmidt-Heydt et al.
(2007). During this work the influence of suble-
thal amounts of the preservatives sodium propi-
onate and potassium sorbate on the expression of
the P. verrucosum ochratoxin polyketide synthase
gene was analyzed. At certain sublethal concen-
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis 367

6 Fig. 17.6. Growth kinetics (top),

otapksPV expression profile
5 (middle) and ochratoxin A-pro-
duction kinetics (bottom) of
Colony growth [cm]

4 P. verrucosum at a changing sin-

gle parameter (water activity).
The major peak of ochratoxin
3
biosynthesis arose at a water ac-
tivity of 0.98, the minor peak at
2 0.93, which completely follows
the respective gene expression
1

0
0.99 0.98 0.95 0.93
100000.00
Gene expression [copy number]

10000.00

1000.00

100.00

10.00

1.00
0.99 0.98 0.95 0.93
2000.00
Toxin biosynthesis{ng/g]

1500.00

1000.00

500.00

0.00
0.99 0.98 0.95 0.93
Water activity

trations of both preservatives the gene was highly
activated, which was paralleled by a higher pro-
duction of ochratoxin A. This induction however
was only observed if the concentration of the
preservative did not exceed a certain level.
When the concentration was too high, growth
was still possible, but ochratoxin production
was reduced. This means that only mild stress,
obviously irrespective of the kind of stress,
induces ochratoxin A biosynthesis, whereas
more extreme conditions leads to an inactivation
of the biosynthesis.
A similar behavior was described for patulin biosynthesis
by P. expansum (Baert et al. 2007). Also, in the case of P.
expansum, limited stress conditions (e.g. low temperature,
low oxygen content) induced patulin biosynthesis, where-
as the application of higher stress factors stopped patulin
biosynthesis. An activation of mycotoxin biosynthesis
genes by stress was also demonstrated for fumonisin pro-
duction in F. verticillioides (Jurado et al. 2008). These
authors found an activation of the fum1 gene by reduction
of the temperature or application of water stress. In con-
trast to the increase in fumonisin biosynthesis, these con-
ditions lead to a reduction in the growth rate, indicating
mild stress for the fungus. In the case of aflatoxin biosyn-
thesis by Aspergillus, it was shown that oxidative stress
368 Rolf Geisen and Markus Schmidt-Heydt
induces toxin biosynthesis (Jayashree and Subramaniam
2000). The aflatoxin production by A. flavus on pistachio
kernel agar can be increased by a factor of about two by
applying oxidative stress by the addition of 100 mM tert-
butyl hydroperoxide (Kim et al. 2006). The addition of a
certain concentration of antioxidants nearly completely
counteract the induction of aflatoxin biosynthesis because
these substances reduce the external oxidative stress. The
authors speculate, because of the fact that the precursors
of aflatoxin are phenolic compounds with predictable
antioxidative activities, that they contribute to the allevia-
tion of the oxidative stress. This counter-action against the
negative effects of oxidative stress by inducing aflatoxin
biosynthesis might be an evolutionary advantage over
species unable to produce the toxin. The same group
(Kim et al. 2005) developed a model system in S. cerevisiae
to analyze the stress response in relation to aflatoxin
biosynthesis. This work identified 43 genes of A. flavus
which are activated during stress responses and which are
possibly linked to aflatoxin biosynthesis.
Because of the fact that obviously several myco-
toxins (aflatoxin, ochratoxin A, patulin, trichothe-
cenes, fumonisins) are activated under stress
conditions, an involvement of these mycotoxins
in stress adaptation seems evident. It seems also
likely that the role of the different mycotoxins
during stress adaptation is different. The involve-
ment of aflatoxin in the inhibition of negative
oxidative stress activities seems reasonable. The
biosynthesis of ochratoxin A is activated under a
set of different stresses (probably all stresses),
which indicates a general role in stress adaptation.
Environmental stresses must somehow be
recognized by the cell and the signal must be
transduced to the nucleus to alter gene expression.
Until now not much is known about the connec-
tion between signal transduction pathways, stress
response and mycotoxin gene activation. Recently
Ochiai et al. (2007b) connected the activity of an
osmosensor histidine kinase and other osmotic
stress-activated protein kinases to the regulation
of trichothecene biosynthesis in F. graminearum.
Deletion mutants in several of the kinase genes
(MAPK) resulted in strains unable to produce
trichothecenes, albeit other secondary metabolites
like the red pigment aurofusarin were still pro-
duced. In parallel the expression of two genes of
the trichothecene biosynthesis pathway were
markedly reduced in particular tri4 (a p450-
monooxygenase) and tri6 (a transcription activa-
tor). Interestingly salt stress completely inhibits
toxin biosynthesis in the case of the trichothe-
cenes. This is in sharp contrast to ochratoxin
A biosynthesis in which higher salt concentrations
clearly activate toxin production. This again indi-
cates that the role of various mycotoxins in stress
adaptation seems to be different.
VII. Regulation of Ochratoxin
A Biosynthesis in Penicillium
in Relation to Light
It has long been recognized that light can have a
profound effect on the development, morphology
and secondary metabolite production of various
fungi. Already in 1955 two German scientists de-
scribed the influence of light on mycelium and
conidium formation in Alternaria brassicae
(Witsch and Wagner 1955). Since that time a
wealth of information has been gleaned on this
topic. An important biological feature with this
respect is the molecular clock described in detail
in N. crassa (Dunlap and Loros 2006). A well
studied rhythm also exists in the Zygomycete
Pilobolus. However most details about the molec-
ular components of the circadian clock stems
from N. crassa. Recently a circadian rhythm was
detected in a mycotoxin-producing fungus, in
particular in A. flavus (Greene et al. 2003). A
typical expression behavior was described for cer-
tain genes which were under the control of a
biological clock. However no circadian regulation
of aflatoxin biosynthesis genes was described in
this work, instead an oscillation of the glyceralde-
hyde-3-phosphate dehydrogenase was found. In
addition the development of sclerotia was clock-
regulated. During this work the typical molecular
components of a fungal biological clock was also
identified: these consist of the gene frequency
(frq) which is the endogenous oscillator and
entrains (synchronizes) the clock in relation to
the light/dark period. Two other components are
the white collar1 (WC-1) and white collar2 (WC-
2) proteins. Both proteins can form a dimer which
in turn can regulate downstream genes, the so-
called clock-controlled genes or ccgs. These are
often conidiation-related genes, but the mating-
related genes, glyceraldehyde-3-phospahate dehy-
drogenase, copper methallothionine or trehalose
synthase are also controlled by the clock. This set
of known ccgs suggests that the biological clock of
Neurospora regulates biological functions such as
metabolism and stress responses not related to
conidiation (Lakin-Thomas and Brody 2004).
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis
light
+
WC-1
WC-2
+
frq
Transcription of ccg’s
(clock controlled genes)
e.g OTA genes
Fig. 17.7. Model of the circadian clock from N. crassa according to Lakin-Thomas (2000)
The regulation of ccgs often differs from gene to
gene, so general predictions cannot be made.
The frq gene is regulated by a feedback loop
acting negatively on its own transcription. This
negative feedback is supported by activities of
the WC-1 and WC-2 proteins (Lakin-Thomas
and Brody 2004). A complex of the two WC pro-
teins (white collar complex; WCC) is required
for full transcriptional regulation of frq. The
FRQ protein itself positively regulates the levels
of the WC proteins and negatively regulates the
activity of the WCC. The frq gene has two light
responsive elements (LREs) in its promoter
region, suggesting that light regulation is
mediated via transcription of the frq gene. Also
one of the transcription factors, WC-1, can sense
light at a specific wavelength, as it can act as a blue
light receptor (Lakin-Thomas and Brody 2004). A
simple model of the circadian clock of Neurospora
is shown in Fig. 17.7.
Until now mycotoxin biosynthesis has not
been described to be under circadian regulation.
However first evidence for a circadian regulation
of the ochratoxin A biosynthesis in P. nordicum
and P. verrucosum came from variabilities in the
expression data of ochratoxin A biosynthesis
genes, when the samples were taken at variable
day times. A systematical analysis of this phenom-
enon revealed that the expression kinetics of
ochratoxin A biosynthesis genes (otapks) in P.
nordicum and P. verrucosum exhibit a day/night
oscillation of transcript levels, which was not
the case for any analyzed housekeeping gene
369
-
FRQ
(e.g. b-actin). This oscillation was much more
symmetrical when a constant light/dark rhythm
was ensured. After incubation under constant
dark conditions, the oscillation became rapidly
non-symmetrical and less regulated. This typical
deregulation of the oscillation was also shown for
N. crassa. In contrast to this, the expression of a
housekeeping gene, e.g. the b-actin gene stayed
more or less constant over the observation period.
Figure 17.8 shows the oscillating expression of the
otapks gene of Penicillium.
According to the profile, expression increases
during the night and decreases during daytime.
Similar observations were described by Stinnett
et al. (2007). These authors described that also
in A. parasiticus the production of versicolorin,
a precursor of aflatoxin is higher in the dark
than in the light (Calvo et al. 2004). This is due,
beside the activity of the circadian clock, to a
global regulatory protein velvet A (veA) which is
needed for the activation of secondary metabolite
biosynthesis.
The product of veA is required for expression of aflR and
aflJ two regulatory and activating proteins of the aflatox-
in biosynthesis pathway in A. parasiticus and A. flavus.
This gene is also involved in fungal development in that
it affects cleistothecial development in A. nidulans or
sclerotial development in A. parasiticus (Calvo et al.
2004). Light negatively influences the activity of this
gene, which leads to the reduced formation of cleisthote-
cia and secondary metabolites. A deletion of this gene
leads to a complete loss of transcription of genes involved
in aflatoxin biosynthesis, like the two regulatory-activating
370
300.00
250.00
200.00
Toxin biosynthesis [ng/g]
150.00
100.00
50.00
0.00
6:00
Fig. 17.8. Circadian regulated expression of the otapksPV gene of P. verrucosum. The expression kinetics is a smooth
curve under alternating dark/light conditions, whereas the expression kinetics under constant dark conditions is rapidly
deregulated. Highest expression and biosynthesis was found during the dark
genes aflR and aflJ and the two cluster genes pksL1 and
ver1. This indicates the strong dependence of aflatoxin
biosynthesis on a functional veA gene product. The veA
product is imported into the nucleus by the action of
importin a. This migration to the nucleus is light-depen-
dent. In the dark VEA is located mainly in the nucleus,
whereas in the light VEA can mainly be found in the
cytoplasm (Stinnet et al. 2007). It could also be shown
that various wavelengths of light have different effects.
Blue light most prevents accumulation of VEA in the
nucleus.
Recently homologoues of the veA gene
were identified in P. verrucosum and P. nordicum,
named vePV and vePN. These genes were identified
by heterologous PCR with primers deduced from
A. parasiticus. A PCR fragment from P. verrucosum
or P. nordicum was sequenced and resulted in
sequences homologous to the veA gene of A. para-
siticus, indicating the conservation of this gene.
After inactivation of the gene in P. verrucosum a
phenotype arose which corresponds to the veA–
phenotype of A. parasiticus (Calvo et al. 2004) in
that the P. verrucosum mutant was no longer able
to produce ochratoxin A or citrinin. In addition the
components of the molecular clock could also be
identified by the same approach. Homologues of
the frq and the wc-1 and wc-2 genes could be
identified in P. verrucosum, demonstrating the mo-
lecular basis of a circadian clock in this fungus.
Analysis of the expression kinetics of the frq and
the wc-1 gene demonstrated that this clock is
Rolf Geisen and Markus Schmidt-Heydt
8:00 10:00
12:00 14:00
16:00 18:00 dark
20:00 22:00 light-dark
0:00 2:00
Time [h] 4:00 6:00
functional. Both genes are conversely regulated.
When the frq transcript level is high, the transcript
level of wc-1 is low and vice versa, leading to a
reversed oscillation of transcription of both genes,
as also shown for Neurospora (Dunlap and Loros
2006). Unequivocal evidence that the circadian
clock of Penicillium is involved in ochratoxin A
biosynthesis comes from the fact that a gene-inac-
tivation mutant of the wc-1 gene of P. verrucosum
had a completely altered pattern of ochratoxin A
regulation in response to light. Interestingly and
unexpectedly the mutant produces more ochra-
toxin under light conditions compared to the wild
type. That indicates that WC-1 has a negative ac-
tivity on transcription of ochratoxin A biosynthesis
genes under light conditions whereas vePV has a
positive activity and acts as an antagonist for the
WC complex.
Purschwitz et al. (2008) recently analyzed the physical
interactions between blue and red light sensors in A. nidu-
lans. They found that a phytochrome component (FPHA)
is part of a protein complex containing LREA (WC-1) and
LREB (WC-2). It was shown for A. nidulans that FphA
represses sexual development and mycotoxin (sterigmato-
cystin) formation, whereas LreA and LreB stimulate both.
FPHA seems to interact with LREB and VeA, which was
described already to be involved in light/dark-regulated
mycotoxin biosynthesis. During this analysis it was shown
that blue light repressed mycotoxin biosynthesis to a level
similar to white light, which indicates that the blue light
receptor (LreA) might in addition have a negative
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis
influence on mycotoxin biosynthesis under certain condi-
tions. According to these authors LreA and LreB act posi-
tively and light inhibition on mycotoxin biosynthesis is
mediated by negatively acting FphA.
In Penicillium this regulation seems not be
completely identical, as an inactivation of a wc-1
homologue increases ochratoxin A biosynthesis
drastically. It might be possible that the inactiva-
tion of wc-1 prevents interaction of a putative
FphA protein with the WC complex and thereby
ceases the negative action of the red light receptor,
allowing increased mycotoxin biosynthesis.
Another important hint for differences in light
regulation in Penicillium and Aspergillus comes
from the fact that, although blue light has a nega-
tive impact on the biosynthesis of sterigmatocys-
tin by A. nidulans, this effect is not stronger than
the effect of white light (Purschwitz et al. 2008). In
Penicillium white light has a negative effect on
ochratoxin A biosynthesis too, but blue light
completely blocks mycotoxin biosynthesis or
even growth if applied at higher light intensities.
A similar observation has been made by Hägg-
blom and Unestam (1997). They described that
blue light reduced the production of alternariol
and alternariol-monomethylether by Alternaria
alternata by 69% and 77% respectively. Red light
had no effect on toxin levels. That is similar to the
situation in Penicillium, where it also has been
shown that red light had nearly no reducing effect
on ochratoxin A biosynthesis.
There is another effect of light/dark altera-
tions on the steady-state level of ochratoxin A
produced by Penicillium. It has been stated al-
ready that gene activation and biosynthesis is
increased under dark conditions and reduced dur-
ing light. However, in addition to the drop in
ochratoxin A biosynthesis under light conditions,
the already produced ochratoxin A seems to be
partly degraded. This results under stationary
conditions also in an oscillation of the ochratoxin
A concentration itself, beside the oscillation at the
transcript level. The level of ochratoxin A oscil-
lates around a fixed value. Ochratoxin A is pro-
duced during the night and partly degraded
during the day. The reason for this oscillation is
not completely clear yet. However Gillmann et al.
(1998) showed that the toxin is susceptible to light
and decomposes over time. Their experiments
showed that especially blue light degrades ochra-
toxin A. The same authors however also showed
371
that ochratoxin A has a DNA photocleavage activ-
ity under certain light conditions. So the decrease
in ochratoxin A concentration during the day may
be due to an absorption of the blue light compo-
nents of daylight, thereby protecting the fungus
from these growth-inhibiting wavelengths, or al-
ternatively the ochratoxin A might be actively
degraded by peptidases, which cleave the peptide
bond between phenylalanine and the polyketide
moiety, until an ochratoxin A concentration is
reached, which is no longer toxic for the fungus
under light conditions due to the photocleavage
effect. Stander et al. (2000) demonstrated that
hydrolytic enzyme mixtures containing potential
proteases and amidases are able to cleave ochra-
toxin A in ochratoxin a and phenylalanine. Varga
et al. (2000) demonstrated that different Asper-
gilli, including A. niger, which itself is able to
produce ochratoxin A, can degrade ochratoxin A
to ochratoxin a and phenylalanine. The authors
suggested a carboxypeptidase is responsible for
the degradation. Figure 17.9 summarizes the in-
fluence of light/dark alterations on ochratoxin
biosynthesis by Penicillium.
VIII. Conclusions
Current knowledge of the physiology, molecular
biology and genetics of ochratoxin A biosynthesis
in Penicillia has been described. The reported
results imply that the ochratoxin A biosynthesis
is activated under unfavorable growth conditions
which suppose mild stress conditions to the fun-
gus. Obviously various stress conditions exerted
by different parameters (like temperature, water
activity, pH, suboptimal amounts of preservatives
or fungicides or unfavorable wavelengths of light)
lead to the same biological answer, e.g. the pro-
duction of ochratoxin A to elevated levels. This
suggests that the activation of ochratoxin A
biosynthesis is correlated to stress adaptation
(Fig. 17.10).
The biosynthesis of ochratoxin A might sim-
ply be coregulated with the stress-adaptive re-
sponse without having defined functions in
stress adaptations. However, ochratoxin A might
be a component of the stress adaptation. It is
known that ochratoxin A binds to proteins. For
this reason it may have some kind of protective or
stabilizing functions which increase the activity
372 Rolf Geisen and Markus Schmidt-Heydt

Fig. 17.9. Scheme of the influ-

ence of light/dark rhythms on
ochratoxin A biosynthesis by
Penicillium

Growth parameter

Stress
Light

Signal transduction
Sensor
CREA

PACC
WCC AREA

VEA
Parameter controlled expression
Metabolic/circadian oscillation
Temporal controlled expression

Biosynthesis

Mycotoxin production

Fig. 17.10. Model of the influence between environment and the fungal cell leading to the activation of ochratoxin
biosynthesis genes

window of proteins with respect to enzymatic or
other activities. This of course should lead to an
increased fitness under conditions which lead to
an activation of the ochratoxin A biosynthesis
genes. Indeed a very careful analysis of the growth
rate of ochratoxin A-producing Penicillia under
constantly changing environmental conditions
(for example temperature) reveals two growth
peaks, a major peak of the growth rate at the
well known conditions of 20–25 C (depending
on the strain) and a minor peak at the conditions
which are exactly the mild stress conditions and
which leads to the induction of ochratoxin A bio-
synthesis (for example 15 C). The minor peak can
 Physiological and Molecular Aspects of Ochratoxin A Biosynthesis
only be found after prolonged measurement of the
growth rate. The rate at 15 C at the beginning of
growth is below the rate at higher temperature
(say 20 C) as expected; however after prolonged
incubation the growth rate at 15 C increases and
outruns the rate at higher temperature (20 C).
Very interestingly the acceleration of the growth
rate begins during the growth phase, when
ochratoxin A biosynthesis starts at these mild
stress conditions. Neither of the several non-
ochratoxin A-producing Penicillium species test-
ed showed this behavior, indicating that ochra-
toxin A biosynthesis and acceleration of growth
rate is coupled. Similar increases in growth rate
after onset of ochratoxin A biosynthesis were ob-
served after changing other parameters like salt
concentration, indicating that this might be a gen-
eral phenomenon.
It is tempting to speculate according to these
recent results, that the biosynthesis of ochratoxin
A might increase the fitness of the fungus under
mild stress conditions, like low temperature, a
certain salt concentration or other parameters.
What is clear at this point is the fact that under
mild stress conditions ochratoxin A biosynthesis
increases and at the same time the growth rate
accelerates in a later growth phase when ochra-
toxin A is produced. However it is not clear at this
stage whether the activation of ochratoxin A bio-
synthesis is the cause or consequence of growth
acceleration. Further research in connecting stress
responses and ochratoxin A biosynthesis at the
molecular level is needed to understand this obvi-
ous link.
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18 Genetic and Metabolic Engineering in Filamentous Fungi

JOCHEN SCHMID1, ULF STAHL1, VERA MEYER1,2

CONTENTS mutagenesis techniques. The development of

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 recombinant DNA technologies for filamentous
II. Fungal Genomics: Advances fungi, shown for the first time in 1979 for Neuros-
in Exploring Sequence Data . . . . . . . . . . . . . . . . . . 378 pora crassa, was a milestone in obtaining insights
III. Post-Genomic Approaches to Unravel into the molecular basis of product formation and
the Metabolism of Filamentous Fungi . . . . . . 379
A. Transcriptomics . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 to improve traditional fungal fermentations by
B. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 genetic engineering.
C. Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 The industrial relevance of filamentous fungi
IV. Metabolic Engineering: Finding the is based on their high capacity to produce primary
Optimum Genetic Strategy . . . . . . . . . . . . . . . . . . . 381 and secondary metabolites as well as for secreting
A. Choosing the Right
Transformation Technique . . . . . . . . . . . . . . . . . . . 382 proteins, having a wide spectrum of activity
V. Enhancing Gene-Targeting Efficiency . . . . . . . 383 such as hydrolases and proteases (Conesa 2001;
A. RNA-Based Tools for Metabolic Engineering 383 Punt et al. 2002). Additionally, the fungal glyco-
VI. Concluding Remarks and Prospects . . . . . . . . . 386 sylation machinery is capable of providing a more
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386 ‘mammalian-like’ glycosylation pattern to pro-
teins compared to the commonly used yeast
hosts (Karnaukhova et al. 2007; Nevalainen et al.
I. Introduction 2005; Ward et al. 2004), making filamentous fungi
very attractive for the production of proteins used
The groundwork for modern fungal biotechnology in medical applications. Secretion is, in particular,
was laid in the beginning of the twentieth century, related to fungal morphology as it is thought to
accompanied by advances in microbiology, bio- take place at the growing fungal tip (Fischer et al.
chemistry and fermentation technology. The pio- 2008; Khalaj et al. 2001; Torralba et al. 1998).
neering works of Jokichi Takamine (production of One focus of current research is thus on fungal
amylase from koji mold Aspergillus oryzae, 1894), morphology to improve the secretory capacity of
James Currie (development of fungal fermentation industrially used fungi (Grimm et al. 2005; Meyer
for citric acid production, 1917) and Alexander et al. 2008; Papagianni et al. 2003).
Fleming (discovery of penicillin production by The following three sections are devoted to
Penicillium notatum, 1928) stimulated scientists three important areas of research of genetic engi-
to further explore fungal metabolic capacities neering in filamentous fungi, all aiming at the
and, moreover, prompted engineers to develop improved application of these organisms in bio-
large-scale and controlled production processes technology.
for filamentous fungi. Improvements of fungal Section II summarizes current genomic
capacities to produce metabolites of interest approaches for filamentous fungi and discusses
were, however, mainly restricted to classic their benefit for the identification of new commer-
cially interesting products.
Section III highlights the progress made in the
1
University of Technology Berlin, Department of Microbiology post-genomic era, concerning new omic techni-
and Genetics, Gustav-Meyer-Allee 25, 13355 Berlin, Germany; ques as well as related challenges and future per-
e-mail: Ulf.Stahl@lb.tu-berlin.de, j.schmid@lb.tu-berlin.de spectives.
2
Leiden University, Institute of Biology, Research Group for
Section IV deals with genetic and metabolic
Molecular Microbiology, Wassenaarseweg 64, 2333 AL Leiden,
The Netherlands
engineering tools applicable nowadays in

Physiology and Genetics, 1st Edition

The Mycota XV
T. Anke and D. Weber (Eds.)
© Springer‐Verlag Berlin Heidelberg 2009
378 Jochen Schmid et al.
filamentous fungi and highlights the progress
made for different transformation techniques
and gene knockout/knockdown approaches.
II. Fungal Genomics: Advances in
Exploring Sequence Data
Identification of putative new and industrial appli-
cable enzymes or secondary metabolites, e.g. of
medical interest, requires evaluation of the genetic
potential of a given organism. Sequence data has to
be screened for genes coding for desired enzymes
involved in the pathway of interest. To meet the
demand for sequence information, more and more
fungal genomes have been sequenced since the first
genome of the yeast model organism Saccharomyces
cerevisiae was published (Goffeau et al. 1996).
Generally, genome sequencing projects con-
tinue to unravel the genetic capabilities of many
organisms, resulting in more than 800 fully
sequenced genomes currently being available, 25
of which are fungal genomes. More than 2500
sequencing projects are ongoing, including more
than 300 fungal projects (http://www.genomeson
line.org/gold_statistics.htm), whereby industrial
and medically interesting fungi are mainly in the
focus of genome sequencing projects (Table 18.1).
These genomes have been partially or completely
sequenced, or they are currently being re-sequenced. How-
ever, even fully sequenced genomes such as for S. cerevisiae
are still undergoing correction with regard to sequence
details, splicing or improved annotation (Jones 2007).
Comparative genomics is a very useful tool to compare
less well-studied species with better understood model
organisms and to clarify hypothetical genes by computa-
tional annotation. Furthermore, they help to identify dif-
ferences between closely related species as regards
pathogenicity, secondary metabolism or other properties
(reviewed by Jones 2007). For example, the genome of
Trichoderma reesei is considered to be a promising candi-
date for biofuel production from cellulose fibers. It should
eventually become a monosaccharide-producing factory
through the use of rational metabolic engineering (Marti-
nez et al. 2008).
‘Gene mining’ as a tool for unravelling new pro-
teins masked in fungal genomes is an approach
which focuses on the industrial exploitation of
currently unknown proteins and enzymes. For ex-
ample, over 200 new proteases have been identified
during annotation of the A. niger genome, of
which two have already been commercialized –
a protease preventing chill-haze in beer
(Lopez and Edens 2005) and a protease used for
the production of sport drinks (Edens et al. 2005).
Detailed analysis of the A. niger genome combined with
the reconstruction of the metabolic networks identified
many gene duplications, transporters and intra- and ex-
tracellular enzymes, providing new insights into the effi-
cient citric acid production by A. niger (Pel et al. 2007).
The A. oryzae genome was found to be extremely rich in
genes involved in biomass degradation and primary and
secondary metabolism (Abe et al. 2006; Kobayashi et al.
2007). New a-amylases and a-glucosidases were found.
The genome sequence revealed 134 peptidase genes, in
contrast to 18 peptidases hitherto known (Kobayashi
et al. 2007). Comparative genomics led to improved func-
tional annotation of the genome of A. nidulans and deeper
insights in pathway modelling (David et al. 2008).
Furthermore, the so-called ‘genome-based strain
reconstruction’, based on a comparison of high-
producing strains with the wild-type strain, led to
reconstructed strains superior to former produc-
tion strains (Adrio and Demain 2006; Ohnishi
et al. 2002).
An absolute novelty decrypted by genome
data and first metabolic approaches was the iden-
tification of (silent) gene clusters for mycotoxin
production in fungal strains which have been
regarded to be safe for many years. Species such
as A. oryzae, A. niger and A. sojae were shown to
produce or have the opportunity of mycotoxin
synthesis dozing in the genome (Jones 2007; Pel
et al. 2007). Dependent on sequencing results,
these facts indicate that using these fungi (and
other species) could be more risky than initially
thought and might thus require new risk assess-
ments (Abe et al. 2006).
‘Metagenomics’ or ‘environmental sequenc-
ing’ involves the sequencing of whole ecosystems,
independent of specific organisms. This boosts
the amount of sequence data available (Raes
et al. 2007). Metagenomics can be used to identify
and analyze fungal communities (O’Brien et al.
2005), explicitly for chosen proteins (mostly poly-
saccharide-modifying enzymes, proteases, nitri-
lases; Blackwood et al. 2007; Streit and Sehmitz
2004), or to sequence fungi which cannot yet be
grown in culture and therefore cannot be analyzed
by traditional means. Environmental sequencing
will play a major role in the decryption of complex
communities and synergisms among different
organisms in their natural habitats.
‘Functional sequencing’ allows insight into the
real natural behavior over and above the limiting
 Genetic and Metabolic Engineering in Filamentous Fungi 379

Table 18.1 Genome sequencing status of selected filamentous fungi

Organism Strain Remark Relevance URL

Aspergillus clavatus NRRL 1 Annotated Medical http://www.tigr.org/tdb/e2k1/acla1/
A. flavus NRRL 3357 Annotated Medical http://www.Aspergillusflavus.org/
genomics/
A. fumigatus Af293 Annotated Medical http://www.tigr.org/tdb/e2k1/afu1/intro.
shtml
A. nidulans FGSC A4 Annotated Model organism http://www.broad.mit.edu/annotation/
genome/Aspergillus_nidulans/Home.
html
A. niger ATCC 1015 Annotated Industrial http://genome.jgi-psf.org/Aspni1/Aspni1.
home.html
A. niger CBS 513.88 Annotated Industrial http://www.dsm.com/en_US/html/dfs/
genomics_aniger.htm
A. oryzae RIB40 Annotated Industrial http://www.bio.nite.go.jp/dogan/
MicroTop?GENOME_ID=ao
A. terreus NIH 2624 Annotated Medical http://www.broad.mit.edu/annotation/
genome/Aspergillus_terreus/Home.
html
Coprinus cinereus 7#130 Annotated Model organism http://www.broad.mit.edu/annotation/
genome/Coprinus_cinereus/Home.
html
Fusarium oxysporum FGSC 4286 Annotated Plant pathogen http://www.broad.mit.edu/annotation/
genome/fusarium_group/MultiHome.
html
Laccaria bicolor S238N-H82 Annotated Symbiotic http://genome.jgi-psf.org/Lacbi1/Lacbi1.
home.html
Magnaporthe grisea 70-15 Annotated Plant pathogen http://www.broad.mit.edu/annotation/
genome/magnaporthe_grisea/Home.
html
Neosatorya fisheri NRRL 181 Annotated Medical http://www.tigr.org/tdb/e2k1/nfa1/
Neurospora crassa OR74A Annotated Model organism http://www.broad.mit.edu/annotation/
genome/Neurospora/Home.html
Phanerochaete RP-78 Draft assembly Industrial http://genome.jgi-psf.org/whiterot1/
chrysosporium whiterot1.home.html
Sclerotinia sclerotiorum 1980 Annotated Plant pathogen http://www.broad.mit.edu/annotation/
genome/sclerotinia_sclerotiorum/
Home.html
Trichoderma reesei QM6a Annotated Industrial http://genome.jgi-psf.org/Trire2/Trire2.
home.html
Ustilago maydis 521 Annotated Plant pathogen http://www.broad.mit.edu/annotation/
genome/ustilago_maydis/Home.html

conditions of cultivating strains under laboratory
conditions and provides excellent opportunities
to identify new and as yet unknown proteins and
enzymes.
III. Post-Genomic Approaches
to Unravel the Metabolism
of Filamentous Fungi
New ‘omic’ tools allow fast and easy decryption of
fungal genomes and the metabolites produced.
These tools can be used to explore new commer-
cially interesting products and to improve meta-
bolic fluxes by rational design. The main
characteristic of the so-called ‘post-genomic’ era
is the need for methods and tools to analyze the
huge amount of data produced. The suffix ‘omics’
was created in the 1990s in the field of bioinfor-
matics and marks the realization of the impor-
tance of information processing in biology;
omics is a general term for a broad discipline of
science and engineering analyzing the interaction
of biological information in various ‘omes’. These
include the genome, transcriptome, proteome,
380 Jochen Schmid et al.
metabolome, expressome, interactome and many
more defined fields, such as glycome, ionome,
lipidome or even physiome and reactome.
In most cases, genomics, transcriptomics and proteomics
together with metabolomics are used to identify theoreti-
cal knockout events or metabolic fluxes in pathways of
interest. The main focus is on gathering information for
engineering metabolic networks to manipulate the reg-
ulatory mechanisms of the entirety of biological processes.
The term ‘systems biology’ is often used in com-
bination with omics and is the ‘biology’ that fo-
cuses on complex systems in life. It is a holistic
approach which allows analysis of the topology of
biochemical and signalling networks involved in
cellular responses; in addition it is able to capture
the dynamics of the response. While different
omics deliver only a piece of the puzzle, it is
hoped that systems biology will eventually map
the complete picture (Siliang Zhang 2006).
A. Transcriptomics
RNA-based technologies have been developed
enormously in the past few years, whereby two
different strategies have emerged: (i) strategies
based on the knowledge of DNA sequences
(microarrays) and (ii) strategies which do not
require any sequence information, such as sup-
pression subtractive hybridization (SSH) and
serial analysis of gene expression (SAGE Break-
spear and Momany 2007). The first 50 fungal
microarrays were reviewed by Breakspear and
Momany (2007), describing the development of
fungal array approaches. Interestingly, they high-
light the lack of basidiomycete microarray experi-
ments (only three have been performed to date).
Comparative transcriptional analysis of
A. oryzae using DNA microarrays indicated the
potential of new proteins identified; and it is as a
tool which can be used to develop industrial sys-
tems (Abe et al. 2006). Anderson et al. (2008b)
used a comparative transcriptomic approach
where one Gene Chip was developed for transcrip-
tome analysis of triplicate batch cultivations of
A. nidulans, A. niger and A. oryzae.
They were able to identify 23 genes conserved across
Aspergillus spp. (mainly sugar transporters and enzymes)
and 365 genes which were differently expressed in only
two of the Aspergilli. Thus, such a cross-species study
based on transcriptome analysis can be used for the
identification of new enzymes or even strains. In a former
publication the authors described the use of transcriptome
data to perform metabolic network modelling in A. niger,
which is explained in detail in Sect. III.C (Andersen et al.
2008).
SSH and SAGE approaches also helped to under-
stand the infection process of plant pathogen
fungi and mycoparasitic fungi. (Carpenter et al.
2005; Gowda et al. 2006; Larraya et al. 2005;
Matsumura et al. 2005; Salvianti et al. 2008;
Schulze Gronover et al. 2004; Yu et al. 2008).
Furthermore, transcriptomic analysis of different
culture conditions will help to identify genes
involved in product formation and may lead to
overproducing strains which are ‘debugged’ with
respect to bottlenecks or unnecessary genes
(Foreman et al. 2003). Also, insights in energy
and primary metabolism have been achieved by
transcriptional analysis (Maeda et al. 2004). The
method of transcriptional profiling has also
been used to reveal previously unknown, but
relevant pathways (Rautio et al. 2006; van der
Werf 2005).
B. Proteomics
Transcriptomic approaches are mostly based on
the straightforward development of microarray
chips, whereas proteomic fields require analytical
techniques such as mass spectrometry (MS) or
nuclear magnetic field resonance. The latest
review of Aspergillus proteomics provides a sum-
mary of all recent information on this technique
(Kim et al. 2008).
In 10 different studies using Aspergillus proteomics, a total
of 28 cell surface proteins, 102 secreted and 139 intracellu-
lar proteins were identified. A proteome map highlighting
proteins identified in major metabolic pathway is summar-
ized by Kim et al. (2008).
For Phanerochaete chrysosporium, more than
1100 intracellular and 300 mitochondrial proteins
were resolved in 2D gels and the metabolic flux
shift was determined by comparative proteomics
for growth on vanillin. By using vanillin, which is
the decomposition product of lignin, the lignin
degradation by white rot fungi can be studied.
Many more proteomic approaches have been per-
formed using fungi to identify new products for
industrial applications, as recently reviewed by
Kim et al. (2007).
 Genetic and Metabolic Engineering in Filamentous Fungi
C. Metabolomics
It is assumed that around 10 000 secondary meta-
bolites may be produced by fungi, examples of
which are found in Aspergillus and Penicillium;
however, less than 10% are known (Smedsgaard
and Nielsen 2005). Much understanding of the
central metabolism and regulation in less-studied
filamentous fungi can be learned from compara-
tive metabolite profiling and the metabolomics of
yeast and filamentous fungi (Smedsgaard and
Nielsen 2005). Metabolomics is considered to be
more sensitive than transcriptomics or proteo-
mics due to the measuring of metabolite concen-
trations caused by environmental perturbations
that may not affect transcription or protein levels,
an example being enzyme activity levels (Oliver
et al. 1998). The use of metabolomics means that
methods are available that can simultaneously
determine over 1000 charged metabolites using
capillary electrophoresis-MS (Soga 2007). Even
techniques which simulate the whole cell metabo-
lism have been developed (Ishii et al. 2004).
A. nidulans represents an important model organism for
studies of cell biology and gene regulation. Kouskoumve-
kaki et al. (2008) initiated a metabolomics approach in
recombinant Aspergilli for clustering and classification.
More than 450 detected metabolites were analyzed and
resulted in the identification of seven putative biomarkers
by which classification into genotype was possible. Thus,
metabolite profiling is a powerful tool for the classification
of filamentous fungi as well as for the identification of
targets for metabolic engineering (Kouskoumvekaki et al.
2008).
A metabolic model integration of the bibliome,
genome, metabolome and reactome was con-
structed for A. niger (Andersen et al. 2008).
A complete metabolic network was presented
which shows great potential for expanding the
use of A. niger as a high-yield production platform.
By the multi-omic approach, the use of precursors
was identified to increase productivity. In order to
combine the knowledge of the underlying meta-
bolic system and the analysis of the data, novel
computational approaches and methods that go
beyond commonly used statistical techniques are
required (Van Dien and Schilling 2006).
Progress has been made using methods for analyzing
quantitative metabolomics data in the context of the
entire network. Methods based on Gibbs energies of for-
mation, the second law of thermodynamics and on known
381
direction reactions within the cell allow dynamic analysis
of metabolites in the cell (Kummel et al. 2006).
Mainly, metabolic flux analysis (MFA) has become
a fundamental tool in metabolic engineering to
elucidate the metabolic state of the cell and has
been applied to various biotechnological processes
(Spegel et al. 2007). ‘Conventional MFA’ is based
on mass balances using stoichiometric constraints
coupled to extracellular product formation rates
and is a widely used technique (Vallino and Ste-
phanopoulos 2000).
Applying the full range of omics technologies
for strain improvement, it would be desirable if
the ‘concept of sample’ was shared among these
technologies (in particular, to focus on a
biological sample that is prepared for use in a
specific omics assay; Morrison et al. 2006). A
common data file format should be found to en-
able fast and secure file sharing and to reduce
redundant data and the potential for errors. The
main issue as regards combining the analysis of
metabolomics data with other omics results is the
data integration problem (Mendes 2006).
There is also the call for standards in sample
collection and processing to obtain reliable results
and to avoid significant error in omics data. It is
essential to use the same biological sample when
confronted with diverse omic approaches
(Martins et al. 2007; Weckwerth et al. 2004).
This has resulted in many omics standardization initia-
tives aiming at the development of new concepts to over-
come the problems of data confusion and sample
correlation (Jones et al. 2007; Morrison et al. 2006).
Also claimed is the necessity to develop common data-
bases for storage of metabolomics data (Kouskoumvekaki
et al. 2008).
IV. Metabolic Engineering: Finding
the Optimum Genetic Strategy
For successful optimization of a fungal metabo-
lism, several issues of engineering strategy have to
be taken into account. Every approach depends on
the organism of choice, the target to be engineered
and the aim of the intervention. From various
genetic engineering tools available nowadays for
filamentous fungi, the most suitable has to be
chosen in order to specifically and efficiently
improve the strain of interest.
382 Jochen Schmid et al.
A. Choosing the Right Transformation
Technique
Highly sophisticated genetic methods are avail-
able nowadays for filamentous fungi; however,
safe and suitable transformation techniques are
fundamental prerequisites for genetic engineering
approaches. Different transformation techniques
enable scientists to design and develop rational
metabolic engineering strategies for industrially
important fungal species. Linear DNA or plasmids
are transferred into the fungal cell, either by
chemical treatment of protoplasts using Ca2+ and
polyethylene glycol (PEG; Spegel et al. 2007), by
physical treatment such as electroporation and
biolistic procedures or by using Agrobacterium
tumefaciens-mediated transformation (AMT;
Casas-Flores et al. 2004; Meyer 2008; Michielse
2005; Ruiz-Diez 2002; reviewed by Fincham 1989).
Historically, PEG-mediated transformation of protoplasts
was the first transformation technique established in 1978
for the budding yeast S. cerevisiae (Hinnen et al. 1978).
This method was afterwards used in filamentous fungi
(Fincham 1989; Punt et al. 1987; Ruiz-Diez 2002; Stahl
1987). Progress in the past few decades was achieved in
establishing alternative transformation methods to over-
come the limits related to protoplast formation, which is
highly dependent on the quality of the cell wall-degrading
enzyme preparation and often only leads to low transfor-
mation rates (Michielse 2005).
In most cases, asexual spores such as conidia or
sporangiospores are used as they are considered
to be the most favorable constituent for transfor-
mation. However, if these are not available, the
whole mycelium has to be transformed, making
the procedure more difficult and laborious. In
addition, the multinuclear state of some conidia,
mycelia and protoplasts hampers the selection of
transformants and makes the selection process
tedious (Deed 1989; Farina et al. 2004). When
comparing the transformation rates of yeast and
filamentous fungi, it becomes evident that trans-
formation of yeast is usually much more efficient
(Ruiz-Diez 2002).
New systems such as electroporation have
been developed. This is actually a method often
used for the transformation of filamentous fungi
(reviewed by Ruiz-Diez 2002). There are three
possible fungal structures that can be transformed
by means of electroporation: protoplasts, conidia
or young germlings (Chakraborty et al. 1991;
Ruiz-Diez 2002).
Biolistic transformation was introduced in
1987 (Klein et al. 1992; Sanford 1987) and has
been applied to a number of filamentous fungi
(Ruiz-Diez 2002). This method is especially bene-
ficial for those organisms which cannot be trans-
formed by AMT. The use of A. tumefaciens as a
mediator of foreign DNA into fungal cells is a tool
which has been adapted for many different fungal
organisms in the past few years (Michielse 2005).
The transformation rate is up to 100–1000 times
higher than with protoplastation and can be used
for most fungi which cannot be transformed by
other methods or where protoplasts do not regen-
erate sufficiently (Casas-Flores et al. 2004; MacK-
enzie 2004; Meyer et al. 2003). However, even this
method is not suitable for all fungi. There are also
reports on less successful attempts, for example in
A. niger (Michielse 2005).
AMT and all other transformation techniques cannot be
used for every fungal organism without first being
adapted. This may be explained by major differences
found in fungal genomes such as A. nidulans (Galagan
et al. 2005), A. oryzae (Machida et al. 2005) and A. fumi-
gatus (Nierman et al. 2005). Analysis of their genomes
shows that there is only 68% of amino acids identity
shared by all three species (Galagan et al. 2005), an evolu-
tionary distance comparable to that between human and
fish (Dujon et al. 2004; Fedorova et al. 2008). Roughly 70%
of A. nidulans genes could be mapped to a syntenic block
with either A. fumigatus or A. oryzae, with about 50% of A.
nidulans in conserved synteny across all three species
(Galagan et al. 2005), suggesting that this could be one
reason for the observation that each transformation meth-
od has to be adapted and optimized for every single spe-
cies and even strain.
Apart from transformation approaches, sexual
crossing is a useful tool to improve the pheno-
type of filamentous fungi. Unfortunately, most
medically or industrially interesting fungi lack
a sexual cycle, precluding them from classic ge-
netics. However, insights into the genome of
A. fumigatus and A. oryzae revealed that both
are potentially capable for sexual reproduction
(Dyer and Paoletti 2005; Galagan et al. 2005;
Paoletti et al. 2005), suggesting that classic
genetic tools can also be established for these
fungi. These findings challenge the whole taxon
of fungi imperfecti and even highlight the power
of genome analysis for taxonomical (re-)classifi-
cation.
A very important and interesting question as
regards the different transformation systems is
 Genetic and Metabolic Engineering in Filamentous Fungi
the fate of the introduced DNA. DNA which has
been introduced will be either maintained auton-
omously (occurs rarely) or integrated into the
genome via homologous or heterologous recom-
bination. Homologous recombination targets the
foreign DNA to regions showing sufficient homol-
ogy, whereby the DNA becomes integrated into
the genome as a single or tandem copy. In con-
trast, heterologous recombination events occur
randomly, resulting in single or multi-copy
integrations (de Groot et al. 1998; Malonek and
Meinhardt 2001; Mullins and Kang 2001).
In the case of AMT, mainly single-copy integration events
occur, whereas mainly multi-copy integrations are ob-
served after transformation by PMT. This observation
can have an important impact on the choice of a suitable
transformation system for the design of a metabolic engi-
neering strategy. AMT can be the method of choice for
targeted integration into the genome and PMT can be used
for ectopic and multi-copy integration to improve protein
expression in the strain of interest (Meyer 2008). The latter
strategy has indeed been shown to be a powerful tool for
protein overexpression in Aspergillus and Trichoderma
(Askolin et al. 2001; Lee et al. 1998; Verdoes JC 1995).
In addition, it was found that AMT can increase
homologous recombination frequency, for example
in A. awamori (Michielse et al. 2005). This obser-
vation pointed to new application opportunities of
AMT as a suitable method for directed and inser-
tional mutagenesis (Betts et al. 2007; Lee and
Bostock 2006; Michielse 2005; Sugui et al. 2005).
V. Enhancing Gene-Targeting Efficiency
Metabolic engineering can be used to delete genes
of unwanted side-pathways, thereby redirecting
the metabolic flux to the required product-
forming pathway. In addition, targeted integra-
tion of genes to a genomic locus known to
strengthen transcription is one strategy to en-
hance protein expression and thereby to improve
productivity of an industrial process. Besides,
gene targeting is also the method used for func-
tional genomics. The mode of integration of for-
eign DNA is determined by two competing
processes – homologous recombination (HR)
and the non-homologous end-joining (NHEJ)
pathway (Dudasova et al. 2004; Krogh and
Symington 2004). The low rates of HR events
usually observed can render some filamentous
383
fungi unattractive for many industrial applica-
tions.
However, advances in fungal gene targeting
were recently achieved by suppressing the NHEJ
pathway (for a review, see Meyer 2008). As sum-
marized in Table 18.2, a dramatic increase in HR
efficiency was reported when strains were used in
which the NHEJ pathway was inactivated (Goins
et al. 2006; Ishibashi et al. 2006; Kooistra et al.
2004; Krappmann et al. 2006; Meyer et al. 2007;
Nayak et al. 2006; Ninomiya et al. 2004; Poggeler
and Kuck 2006; Takahashi et al. 2006).
Phenotypic analysis of defective NHEJ strains
revealed that these strains showed higher sensi-
tivity to various toxins and irradiation (da Silva
Ferreira et al. 2006; Meyer et al. 2007; Ninomiya
et al. 2004). Therefore, some unexpected growth
behavior could appear, and the more elegant solu-
tion would be a transient silencing of the NHEJ
pathway, as recently reported for Candida glab-
rata and A. nidulans (Nielsen et al. 2008; Ueno
et al. 2007).
A. RNA-Based Tools for Metabolic Engineering
RNA technology is an attractive alternative to
DNA-based methods to silence gene expression
post-transcriptionally and thereby to control
unwanted metabolic pathways. Different RNA
techniques such as antisense RNA, RNAi and
hammerhead ribozymes have been shown to be
valuable tools for filamentous fungi (Table 18.2).
These methods provide the advantage of not de-
leting a gene, thereby bypassing the possibility of
lethal or other unwanted effects on the organism.
RNA-based methods are especially valuable
when: (i) gene-targeting approaches fail, (ii) mul-
tiple copies of a gene of interest are present in the
genome or (iii) isogenes might compensate for the
knockout of the deleted gene (Akashi et al. 2005).
For example, silencing a whole gene family using
only a single antisense RNA construct has been
described for A. oryzae (Yamada et al. 2007). An-
other advantage of the RNAi mechanism is its
locus independence due to its mediation by a
mobile trans-acting signal in the cytoplasm. Con-
sequently, this mechanisms can be used in fungi
which have multi-nuclear hyphae or a low target-
ing efficiency, even in heterokaryotic fungal
strains, RNA-based downregulation of genes is
possible (de Jong et al. 2006; Nakayashiki 2005).
384 Jochen Schmid et al.

Table 18.2. Genetic tools applicable to filamentous fungi for gene targeting and silencing

Organism References Efficiency

Aspergillus awamori
Antisense RNA Moralejo et al. (2002), Lombrana et al. (2004) 80%
Aspergillus flavus
RNAi McDonald et al. (2005) <90%
Improved gene targeting Chang (2008) 96%
Aspergillus fumigatus
RNAi Bromley et al. (2006), Henry et al. (2007) 80%
Improved gene targeting da Silva Ferreira et al. (2006), Krappmann et al. (2006) 75–96%
Aspergillus giganteus
Ribozyme Mueller et al. (2006) 20–100%
Aspergillus nidulans
RNAi Khatri and Rajam (2007) 40%
Antisense RNA Bautista et al. (2000), Hoffmann et al. (2000), >50–90%
Herrmann et al. (2006)
Improved gene targeting Hammond and Keller (2005) 90%
Aspergillus niger
RNAi Nayak et al. (2006), Barnes et al. (2008) 21–82%
Antisense RNA Ngiam et al. (2000) 60–70%
Improved gene targeting Meyer et al. (2007), Barnes et al. (2008) 80%
Aspergillus oryzae
RNAi Yamada et al. (2007) 90%
Antisense RNA Zheng et al. (1998), Kitamoto et al. (1999) 75–80%
Improved gene targeting 63–87%
Aspergillus parasiticus
RNAi McDonald et al. (2005) 90%
Improved gene targeting Chang (2008) 96%
Aspergillus sojae
Improved gene targeting Kadotani et al. (2003), Nakayashiki (2005) 64–75%
Coprinopsis cinereus
RNAi Walti et al. (2006) 90%
Cryphonectria parasitica
Improved gene targeting Lan et al. (2008) 80–96%
Fusarium solani
RNAi Ha et al. (2006) >98%
Magnaporthe oryzae
RNAi Kadotani et al. (2003), Nakayashiki et al. (2005), 70–90%
Caracuel-Rios and Talbot (2008)
Antisense RNA Kadotani et al. (2003), Nguyen et al. (2008)
Improved gene targeting Villalba et al. (2008) >80%
Neurospora crassa
RNAi Cogoni et al. (1996), Goldoni et al. (2004), 11–49%
Choudhary et al. (2007)
Antisense RNA Tentler et al. (1997), Kramer et al. (2003), Fecke (2008) 50–80%
Improved gene targeting Ishibashi et al. (2006) 91–100%
Penicillium expansum
RNAi Schumann and Hertweck (2007) 95%
Antisense RNA Garcia-Rico et al. (2007) 95%
Podospora anserina
RNAi El-Khoury et al. (2008) 100%
Schizophyllum commune
RNAi de Jong et al. (2006) 80%
Trichoderma harzianum
RNAi Cardoza et al. (2007) 71–98%
Trichoderma reseei
Antisense RNA Wang et al. (2005) 65%
 Genetic and Metabolic Engineering in Filamentous Fungi
Antisense RNA gene silencing is performed by
using single-stranded RNA which is complemen-
tary to an mRNA strand transcribed within the
cell. Formation of a complementary mRNA hybrid
physically blocks the translation machinery and
thereby stops translation of the endogenous
mRNA.
Successful gene silencing using artificial antisense con-
structs have been reported for different filamentous fungi
(Bautista et al. 2000; Blanco and Judelson 2005; Kitamoto
et al. 1999; Lombrana et al. 2004; Ngiam et al. 2000; Zheng
et al. 1998). For example, antisense silencing of the prote-
ase aspergillopepsin B resulted in a reduction of 10–70% of
protease levels and a 30% increase in heterologous thau-
matin production in A. awamori (Moralejo et al. 2002).
However, antisense-mediated reduction of
gene expression to zero levels has not been
reported to date. Nevertheless, exactly this phe-
nomenon can be used for knocking-down gene
expression instead of knocking it out.
For instance, the wide-domain transcription factor CreA,
the key component of carbon catabolite repression in
Aspergillus (Dowzer and Kelly 1991; Ruijter and Visser
1997; Shroff et al. 1997), negatively regulates a number of
industrially important enzymes. Bautista et al. (2000)
reported partial suppression of creA expression in A. nidu-
lans by its antisense molecule (about 50% reduced expres-
sion was estimated) yielding a partial alleviation of glucose
repression and thereby a substantial increase of the pro-
ductivities of intra- and extracellular glucose-repressible
enzymes.
Another RNA-based technology for filamentous
fungi is the use of catalytic RNA molecules, termed
ribozymes. The spliceosome and ribosomes are
two examples for naturally occurring ribozymes.
The so-called hammerhead ribozyme is the smal-
lest and best-studied class of catalytic RNAs
(Akashi et al. 2005; Mueller et al. 2006). The ham-
merhead ribozyme and other ribozymes are anti-
sense RNA molecules. They function by binding to
the target RNA moiety through Watson–Crick base
pairing and inactivate it by cleaving the phospho-
diester backbone at a specific cutting site. The
substrate-recognition arms of hammerhead ribo-
zymes are engineered so that the arms are rendered
complementary to any chosen RNA, enabling
the ribozyme to bind to its target. The functionality
of hammerhead ribozymes as a tool for RNA-based
technology on gene expression has been shown
for bacterial, yeast, plant and mammalian
systems (Akashi et al. 2005; Bussiere et al.
385
2003; Isaacs et al. 2006). Mueller et al. (2006)
recently provided a proof of principle for filamen-
tous fungi.
Congruent to other systems, it has been shown that ribo-
zymes, targeting the 5’ region of a substrate mRNA can
lead to complete gene silencing in A. giganteus, whereas
ribozymes targeting the 3’ region only lead to a partial
reduction (about 20–50%).
RNA interference (RNAi) is a naturally occurring
post-transcriptional gene-silencing phenomenon,
first described in Caenorhabditis elegans and
thereafter in other organisms such as N. crassa
(quelling; Romano and Macino 1992), plants
(‘co-suppression’; Napoli et al. 1990) and animals
(‘RNA interference’; Elbashir et al. 2001a). The
concept of RNAi can be used for artificial gene
silencing in nearly all organisms, even in filamen-
tous fungi. By this method, double-stranded RNA
(dsRNA) trans-genetically delivered to the fungal
interior, is cleaved by Dicer (type-III-ribonucle-
ase) into 21–26 nt small interfering RNA (siRNA).
Dicer is always associated with Argonaute pro-
teins (which bind targeted si/mRNA) and acts by
generating siRNA molecules which in turn target
mRNAs to be silenced. Dicer cleaving products get
incorporated into the ribonucleoprotein complex
(RISC; Bernstein et al. 2001; Hammond et al. 2000).
Homologous mRNAs are subsequently recognized
and degraded via complementary base pairing by
means of incorporated siRNA in the RISC (Elba-
shir et al. 2001b; Zamore et al. 2000). In some
organisms, a RNA-dependent RNA polymerase
(RdRP) can use the antisense siRNA to prime the
conversion of endogenous mRNA into dsRNA
amplifying the silencing signal (Forrest 2004).
The most effective way of post-transcriptional
gene silencing in filamentous fungi can be
achieved using ectopically integrated RNAi con-
structs which usually code for ‘double-stranded
RNA’ molecules. These molecules are self-comple-
mentary hairpin RNAs, formed by an inverted
repeat which is interrupted by a spacer sequence,
and are identical to part of the endogenous se-
quence being targeted (Mouyna et al. 2004). Inser-
tion of an intron in the spacer sequence greatly
increases the silencing efficiency in N. crassa, pos-
sibly due to an enhanced export of the hairpin
from the nucleus during splicing (Goldoni et al.
2004). Remarkably, some fungal strains, e.g.
Ustilago maydis, Candida albicans and S. cerevi-
siae lack components of the RNAi-silencing
386 Jochen Schmid et al.
machinery, indicating that this tool is not applica-
ble for these organisms (Nakayashiki 2005). As
summarized in Table 18.2, specific inhibition of
gene expression by RNAi has been shown to be
suitable for a multitude of filamentous fungi, such
as A. nidulans (Hammond and Keller 2005; Khatri
and Rajam 2007), A. fumigatus (Bromley et al.
2006; Henry et al. 2007), A. oryzae (Yamada et al.
2007), Coprinus cinereus (Namekawa et al. 2005;
Walti et al. 2006), Fusarium solani (Ha et al. 2006),
Magnaportae oryzae (Caracuel-Rios and Talbot
2008; Kadotani et al. 2003; Nakayashiki et al.
2005), N. crassa (Goldoni et al. 2004) and Schizo-
phyllum commune (de Jong et al. 2006). Similar to
antisense strategies, RNAi-induced silencing of
fungal gene expression was most often found to
be incomplete (maximal reduction up to 10% of
wild-type level) and full knockout phenotypes
were seldom observed.
A striking disadvantage of RNAi-based gene silencing is the
instability of the silencing construct and the possibility of
co-silencing unwanted genes (‘off targets’), showing partial
sequence homology to the target gene. Many transformants
lose the RNAi construct after prolonged cultivation.
Chimeric constructs with two genes in tandem
can result in very different silencing efficacies
(Goldoni et al. 2004; Henry et al. 2007; Nakaya-
shiki et al. 2005). In the case of A. fumigatus, it has
been reported that approximately 50% of the
transformants lack the complete RNAi construct
or part of it after prolonged cultivation (Henry
et al. 2007). One possible explanation for this
phenomenon might be the loss of one of the
inverted repeats after the first mitotic event
(Henry et al. 2007).
Still, advances in using RNAi approaches have
been achieved during recent years. For example,
success has been reported by using inducible RNAi
constructs in A. fumigatus (Khalaj et al. 2007) or
by using new uptake methods of artificial siRNA
constructs in A. nidulans (Khatri and Rajam 2007).
Nevertheless, the possibility of off-target effects
can form an obstacle and future work on the
approach has to address the question what is
the optimum sequence length and the minimum
homology to avoid any unwanted co-silencing.
VI. Concluding Remarks and Prospects
The fungal post-genomic era is still in its infancy;
however, the real power of the omic approaches is
the possibility to analyze cellular processes and
responses on different levels, including DNA,
RNA, protein and metabolite levels (Shulaev
et al. 2008).
By integrated analysis of these levels, several
important features of metabolic regulation has
been and will be identified (Le Lay et al. 2006;
Shulaev et al. 2008). Future strategies will combine
all fields of omics and will allow the unravelling of
the dynamics of cellular metabolic activities in
various filamentous fungi. The identification of
new proteins, enzymes, pathways and their
involvement in metabolic networks as well as
‘classic genetic’ techniques and new metabolic
engineering approaches will eventually enable
scientists to develop optimum production strains.
The use of the new and highly sophisticated omic
methods developed in the fungal post-genomic
era will open the floodgates towards high-
throughput analysis and efficient rational meta-
bolic engineering approaches. The knowledge
gained leads to the improvement of industrial
biotechnological processes and will help to meet
the increasing need for sustainable fungal bio-
products. In order to avoid bottlenecks in the
post-genomic era, standardized methods and pro-
tocols have to be established for sampling, sample
processing, sample collection, sample handling
and integration into databanks.
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Biosystematic Index

Absidia glauca, 88 Arthrinum spaeospermum, 285

Acremonium Ascaris suum, 253–257, 259, 261, 266
A. luzulae, 278 Aschersonia, 282
Achlya, 4 Ascochyta, 160, 183–184, 186
Acrostalagmus, 277 Ascomycetes, 81, 85, 87, 89, 135, 306, 309, 316–318, 320
Aegiceras corniculatum, 161, 170–172 Ascomycetous, 81, 87, 88, 94
Agaricales, 12, 13 Ascomycota, 7–9
Agaricomycetes, 12, 13 Ascospores, 19–21, 23, 25, 32–34
Agaricomycetidae, 13 Asellariales, 7
Agaricomycotina, 2, 9, 11–13 Ashbya gossypii, 236
Agaricus, 222 Aspergillus, 9, 91, 93, 204, 249, 251, 257, 259, 380, 381, 383
A. bisporus, 85, 91, 94, 236 A. awamori, 383, 385
Agrobacterium tumefaciens, 382 A. carbonarius, 257, 354, 355, 360–362
Albatrellus confluens, 265 A. carneus, 283
Albugo, 4 A. clavatus, 236, 344, 379
Allomyces macrogynus, 88 A. flavipes, 259
Alternaria, 160, 174–176 A. flavus, 86, 91, 132, 133, 135–137, 141, 144, 300, 314,
A. alternata, 265, 290, 307, 319 379
A. brassicae, 290 A. fumigatus, 48–50, 56, 65, 67, 87, 132, 133, 135–137,
A. citri, 249, 265 140, 141, 143, 144, 236, 274, 288, 298, 300, 307,
A. solani, 338, 340 309, 312, 316, 318, 322, 323, 333, 379, 382, 386
A. tenuis, 249, 265 A. giganteus, 68, 91, 385
Amanita A. nidulans, 48, 50, 51, 67–70, 86, 89, 133, 135–138,
A. bisporigera, 236 142, 143, 145, 146, 236, 298–302, 307, 312, 314–
A. exitialis, 281, 282 316, 318, 320, 324, 378–383, 386
A. muscaria, 89, 92 A. niger, 86, 135, 143, 145, 236, 249, 251, 279, 289,
A. phalloides, 317 354, 355, 371, 378–382
Amoebozoa, 3 A. ochraceus, 353–355, 357, 359–361, 363, 364
Antrodia serialis, 224, 226 A. oryzae, 133, 236, 298, 339, 346, 347, 377–380, 382–
Apiospora montagnei, 259 384, 386
Aporpium caryae, 224, 226, 227 A. parasiticus, 133
Apothecium, 9 A. sojae, 378, 384
Arabidopsis thaliana, 55, 67, 93 A. stellatus, 249
Arachniotus, 277 A. steynii, 354, 355
Archangium gephyra, 263 A. sydowi, 257
Archiascomycetes, 7 A. terreus, 132, 236, 331, 334, 337, 338, 345, 346, 379
Ariolimax columbianus, 95 A. westerdijkiae, 354, 355
Artemisia annua, 161, 187, 188 Assembling the fungal tree of life (AFTOL), 2, 5, 8, 10,
Artemisia maritima, 160, 189 12, 13
Arthoniomycetes, 9 Atheliales, 13
394 Biosystematic Index

Atkinsonella hypoxylon, 199, 201 C. tropicalis, 47, 51, 52, 236

Aureobasidium, 283 Cantharellales, 12, 13
Auricularia, 12 Cassia spectabilis, 162, 166, 167
Auriculariales, 12 Castaniopsis fissa, 280
Avicinnia marina, 280 Catharanthus roseus, 198
Azadirachata indica, 162, 164–165 Caulochytrium, 6
Ceratobasidium, 12
B Ceratocystis, 89
Baccharis artemisioides, 157 C. fagacearum, 86
Baccharis coridifolia, 157 C. ulmi, 86
Bacillus subtilis, 346 Ceriporia subvermispora, 221
Balansia cyperi, 199 Chaetasbolisia erysiophoides, 259
Balansia pilulaeformis, 199 Chaetominum, 277
Basidia, 11, 12 Chaetomium, 160, 176–178
Basidiocarps, 12 C. globosum, 158, 160, 177–178, 236, 344
Basidiomycete(s), 81–85, 87–90, 92, 97, 317–319 C. subaffine, 342
Basidiomycota, 6, 7, 9–13 Chaetothyriales, 9
Basidiospores, 11, 12 Chalara, 279
Batrachochytrium dendrobatidis, 48, 49, 51, 52, 236 Chamonixia pachydermis, 225, 227
Beauveria Chaunopycnis alba, 279
B. bassiana, 67, 68, 140, 141, 144, 276, 340 Chlamydocins, 281, 288
B. fellina, 283 Chlamydomonas reinhardtii, 51
B. nivea, 307, 311 Chrysosporium luchnowense, 212
Beetle(s), 94 Chromista, 4
Bionectria, 277 Chytridiomycota, 5, 6, 88
Bipolaris, 282 Chytrid(s), 4–6
Blastocladiomycota, 6 Cis boleti, 94
Blumeria graminis, 239, 241 Cistanche deserticola, 162, 170–172
Boletales, 12, 13 Classification, 1, 2, 4–6, 8–10, 13
Boletus, 216, 217 Clavariadelphus truncatus, 220
Bondarzewia montana, 224, 226 Clavariopsis aquatica, 289
Boreostereum vibrans, 220 Claviceps, 204, 276, 290
Botryosphaeria mamane, 160, 185 C. puprurea, 315
Botryosphaeria parva, 160, 189 Clavicipitaceae, 198, 199, 202, 205, 278, 322
Botrytis cinerea, 48, 49, 92, 95, 122, 236, 239 Clavicipitaceous fungi, 198–206
Bovista, 212–214 Cleistothecium, 9
Brachypodium sylvaticum, 94 Clitocybe elegans, 217
Burkholderia, 298 Clitocybe faccida, 94
Clitocybe illudens, 213
C Clitopilus prunulus, 94
Caenorhabditis elegans, 385 Clonostachys, 280, 289
Calcarisporium arbuscula, 249, 265 C. lusitaniae, 236
Calocera, 12 Coccidioides immitis, 236
Calonectria Coccidioides posadsii, 236
C. morganii, 280 Cochliobolus
Camellia japonica, 273 C. carbonum, 288, 290
Camellia sinensis L., 161, 165–166 C. heterostrophus, 274
Camptotheca acuminata, 157, 162, 166, 167 C. carbonum, 307, 315, 319
Candida, 7, 73, 241 C. heterostrophus, 332
C. albicans, 67, 69, 84, 86, 91, 110, 123, 236, 239–241, C. miyabeanus, 249, 250
274, 288, 385 Coelomomyces stegomyiae, 51, 52
C. dubliniensis, 236, 239 Coleophoma
C. glabrata, 236, 243, 383 C. crateriformis, 279
C. guilliermondii, 236 C. empetri, 279
C. noadenis, 108 Collembolan, 90, 94
C. parapsilosis, 236 Colletotrichum, 8, 85
 Biosystematic Index 395

C. dematium, 160, 181–182 Dothideomycetidae, 9

C. gloeosporioides, 69–71, 160, 189, 277 Dothistroma septosporum, 335
C. graminicola, 236 Drosophila, 143–147
C. lagenarium, 335, 336, 346 D. melanogaster, 55, 143, 145, 146
C. magna, 155
C. trifolii, 65 E
Coniosporium, 277 Echinococcus multilocularis, 256, 266
Coniothyrium, 160, 189, 276 Ectomycorrhiza, 13
Conocybe siliginea, 211, 212 Embellisia, 206
Convolvulaceae, 197–206 Emericella
Coprini, 83 E. purpurascens, 278
Coprinopsis cinerea, 48, 81–84, 88–91, 95 E. unguis, 280
Coprinopsis cinereus, 69, 70 E. nidulans, 89
Coprinus, 216 E. variecolor, 161, 175, 177, 257
C. cinereus, 236, 379, 386 Endogonales, 6
Cordyceps, 282 Entoloma haastii, 275
Corollospora intermedia, 280 Entomophthorales, 6
Corticiales, 13 Entomophthoromycotina, 6
Cortinarius, 211, 224–226 Entorrhizales, 9, 11
Creolophus cirrhatus, 215, 216 Entorrhizomycetes, 11
Croton oblongifolius, 161, 175 Entylomatales, 11
Cryptococcus aquaticus, 113 Epichloe, 322
Cryptococcus gattii, 236 Epichloe¨ typhina, 94, 161, 181, 280
Cryptococcus humicola, 108 Epicoccum
Cryptococcus neoformans, 46–49, 56, 83, 84, 120, 121, E. nigrum, 278
236, 241, 274 Epinotia tedella, 94
Cunninghamella blakesleeana, 274 Eriospermum, 204
Cyathus, 217 Erysiphales, 9
Cyclindrocladium Erythrina crista-galli, 162, 166–169
C. scorparium, 280 Escherichia coli, 338, 346, 347
Cyclindrotrichum, 278 Euascomycetes, 7
Cylindrocarpon, 280 Euonymus europaeus, 160, 175
Cylindrocladium ilicicola, 249, 262 Eupatorium arnottianum, 169–170
Cystobacter armeniaca, 263 Eupenicillium shearii, 290
Cystobasidiales, 11 Euplotes raikovi, 83
Cystobasidiomycetes, 11 Eurotiales, 9
Cystoderma, 211 Eurotiomycetes, 9
Cystofilobasidiales, 12 Eurotium, 276
Cystofilobasidium infirmominiatum, 113 Exidia, 12
Exobasidiales, 11
D Exobasidiomycetes, 11
Dacrymyces, 12, 216 Exserohilium rostratum, 276
Dacrymycetes, 12 Exserohilum holmi, 277
Debaryomyces hansenii , 47, 50–52, 108, 111, 122, 236
Debaryomyces robertsiae, 113, 117, 119 F
Diaporthe, 160, 164–170 Favolaschia, 218, 219
Dicerandra frutescens, 163, 170, 171 Filobasidiales, 12
Dichomitus squalens, 214, 215 Filobasidiella, 274
Dictyostelium discoideum, 51 Filobasidium capsuligenum, 108
Diheterospora Flavolus arcularius, 92
D. chlamyydosporia, 281 Folsomia candida, 142
Dikarya, 7 Fomitella fraxinea, 219
Dikaryophase, 19 Fungi incertae sedis, 88
Doassansiales, 11 Fungi, 297–299, 301, 302
Dothideomycete, 175–177 Fusarium, 161, 167, 180, 181, 319
Dothideomycetes, 9 F. culmorum, 274
396 Biosystematic Index

F. graminearum, 67, 69, 236, 239, 274 H. mammatum, 155

F. heterosporum, 300 Hormonema dematioides, 161, 182–183
F. lateritium, 205 Hyalodendron, 277
F. moniliforme, 255, 340, 345 Hyaloraphidium, 6
F. oxysporum, 67, 68, 198, 236, 379 Hydnellum caeruleum, 222, 223, 225
F. pseudograminearum, 274 Hygrophorus, 221, 222
F. sambucinum, 249, 262 Hymenochaetales, 13
F. solani, 384, 386 Hymenophore, 12
F. verticillioides, 236 Hyperdermium pulvinatum, 205
Hypericum perforatum, 158
G Hyphochytriomycota, 4
Galleria mellonella, 141 Hypocreaceae, 278
Garcinia mangostana, 160, 184, 185 Hypoderma eucalyptii, 279
Geastrales, 13 Hypodermium bertonii, 205
Gelasinospora cerealis, 91 Hypoxylon fragiforme, 155
Geoglossaceae, 8, 9 Hypoxylon oceanicum, 285
Geoglossum, 8 Hypoxylon truncatum, 161, 188
Georgefischeriales, 11 Hysterangiales, 13
Geotrichum candidum, 274, 289
Geosiphon, 7 I
Gerronema, 222, 223 Idiomorph(s), 20
Gibberella fujikuroi, 132, 133, 331 Imperata cylindrica, 160, 177–178
Gibberella zeae, 89, 274 Ipomoea, 204
Gigaspora, 92 I. asarifolia, 199, 201–204
Ginkgo biloba, 159, 163 I. batatas, 205, 206
Glarea lozoyensis, 280, 337, 345 I. violacea, 199, 202, 203, 205
Gliocladium deliquenscens, 277 Irpex, 218
Gloeophyllales, 13 Isaria, 282
Gloeophyllum, 213, 214
Glomeromycota, 92 J
Glomerella, 8
Junghuhnia nitida, 221
Glomerellaceae, 8
Junipercus cedre, 161, 183–185
Glomospora, 289
Glomus, 92
K
Glomus mossae, 43
Glycine max, 92 Karyogamy, 18, 19
Gomphales, 13 Kickxellales, 7
Grifola frondosa, 219, 220 Kickxellomycotina, 7
Gymnothecia(l), 9 Kluyveromyces, 112
K. lactis, 69, 113, 117–119, 122, 237
H K. marxianus, 237
Haemonchus contortus, 253, 254, 266 K. thermotolerans, 237
Hanseniaspora uvarum, 113, 116, 122 K. waltii, 108, 112, 237
Haptoglossa, 5
Helicobasidiales, 10 L
Helicobasidium, 11 Labyrinthulales, 4
Helicoma ambiens, 280 Labyrinthulomycota, 4
Helminthosporium Lacazia loboi, 237
H. carbonum, 288 Laccaria bicolor, 83, 84, 97, 237, 379
H. siccans, 249, 261 Laccaria laccata, 83
Helotiales, 9 Laetiporus sulfureus, 283
Hemiascomycetes, 7 Lecanoromycetes, 9
Heterobasidiomycetes, 12, 13 Leccinium aurantiacum, 86
Heterobasidion annosum, 237 Leea rubra, 161, 175–176
Hirsutella, 282 Leiodes cinnamomea, 94
Histoplasma capsulatum, 48–50, 56, 85, 237, 240 Lenzites betulinus, 82
 Biosystematic Index 397

Leotiomycetes, 9 Neosartorya fenelliae, 48, 49

Leptographium lundbergii, 213 Neosatorya fisheri, 237, 379
Leptosphaeria, 161, 187 Neotyphodium, 204
L. maculans, 288, 290 Neotyphodium/Epichloe, 289
Leptostroma, 279 Neotyphodium typhinum, 199
Leucopaxillus gentianeus, 219, 220 Nerium oleander, 160, 176–177
Lichen(s), 9 Neurospora crassa, 46, 71, 81, 89, 237, 377, 379, 384–386
Lichen, 344 Nigrosabulum globosum, 284
Limacella illinita, 217 Nodulisporium, 161, 183–185
Littoraria irrorata, 94 Nothapodytes foetida, 198
Lodderomyces elongisporus, 237
Lordithon lunulatus, 94 O
Lower fungi, 5, 7 Olpidium, 6
Olpidium brassicae, 6
M Omphalotus nidiformis, 212, 213
Maackia chinensis, 161, 181 Omphalotus olearius, 212, 213, 281, 317, 318
Macrocystidia cucumis, 214, 215 Onygenales, 9
Magnaporthaceae, 8, 9 Oomycete, 79
Magnaporthe, 8 Oomycota, 4, 5
M. grisea, 73, 237, 238, 241, 243, 274, 314, 316, 318, Orbiliomycetes, 9
320, 333, 340, 344, 379 Oudemansiella mucida, 249, 263
M. oryzae, 346
Malassezia globosa, 237 P
Malassezia restricta, 237 Paecilomyces
Malasseziales, 11 P. cinnamomeus, 283
Marasmius, 214, 215 P. militaris, 259
Medicago lupulina, 162, 183 Paracoccidioides brasiliensis, 48, 49, 121, 237
Megaselia halteria, 94 Parasitella parasitica, 88
Meiosis, 17–20, 25, 28, 29 Paxillus curtisii, 222, 223, 225
Meliotus dentatus, 160, 183–184, 186 Paxillus involutus, 237
Metarhizium anisophilae, 141, 333 Penicillium, 9, 91, 161, 170–174, 249, 258, 333, 381
Metharizium P. chrysogenum, 48, 49, 67, 68, 162, 170–171, 237
M. anisopliae, 277, 289, 290 P. citreoviride, 249, 265
Microbotryales, 11 P. dipodomyis, 276
Microbotryomycetes, 11 P. expansum, 333, 335, 340, 341, 343, 346
Microsporum gypseum, 237 P. fellutanum, 276
Monascus purpureus, 331, 335 P. funiculosum, 249, 257, 262
Moniliophthora perniciosa, 237 P. islandicum, 287
Mortierellales, 6 P. janczewskii, 162, 172, 173, 276
Mucoromycotina, 6, 88 P. lilacinum, 249, 266
Mucorales, 6 P. madriti, 335
Mycena, 212, 213, 217–219, 222, 224 P. marneffei, 237
Mycobacterium tuberculosis, 50, 56 P. nalgiovense, 276, 362, 363
Mycosphaerella graminicola, 239 P. notatum, 87, 377
Mycorrhiza, 7, 13 P. paneum, 85
Mycosphaerella, 94 P. paxilli, 162, 186
M. fijiensis, 237 P. roquefortii, 217
M. graminicola, 237 P. rugulosum, 257
Myriogenospora atramentosa, 199, 201 P. simplicissimum, 276
Myxococcus fulvus, 263 P. vermiculatum, 257
Myxotrichum, 9 P. verrucosum, 354, 355, 357, 361–363, 366, 367, 369, 370
Peniophora, 281, 288
N Perithecium, 9
Nectria haematococca, 89, 237 Peronospora, 4
Neocosmospora vasinfecta, 278 Peronosporales, 4
Neolecta, 7 Peronosporomycetes, 5
398 Biosystematic Index

Pestalotiopsis foedan, 163, 179 Polygonum senegalense, 160, 174–175

Pestalotiopsis theae, 163, 179–180 Polymyxa, 4
Pezizales, 9 Polyporales, 13
Pezizomycetes, 9 Polypores, 94
Pezizomycotina, 7–9 Polyporus dryophilus, 82–83
Phaeosphaeria, 94 Postia placenta, 237
Phaeosphaeria nodorum, 51, 52 Proisotoma minuta, 94
Phanerochaete carnosa, 237 Protomyces, 7
Phallales, 13 Protozoa, 7, 13
Phallomycetidae, 13 Prumnopitys andina, 161, 162, 171–174
Phanerochaete chrysosporium, 48, 237, 314, 315, 317, 318, Psathyrella pseudogracilis, 212
379, 380 Pseudallescheria, 277
Phellinus tremulae, 212 Pseudogymnoascus, 9
Phleum pretense, 161, 181 Pseudomonas, 298
Pholiota spumosa, 225, 227 P. aeruginosa, 91, 262
Phoma Pteromischum, 160, 181–182
P. lingam, 290 Pterula, 222, 224, 250, 283
P. exigua, 333 Puccinia graminis, 85, 237
P. medicaginis, 162, 183 Pucciniales, 10
Phoma sp., 333, 335, 338, 346 Puccinia monoica, 93
Phomopsis, 155, 162, 164–171 Pucciniomycetes, 10
P. asparagi, 347 Pucciniomycotina, 2, 9–11
P. cassiae, 162, 166 Pyrenomycetes, 9
P. longicolla, 163, 170 Pyrenophora triticirepentis, 48, 49, 237
Phorid fly, 94 Pyrenulales, 9
Phycomyces blakesleeanus, 237 Pyronema domesticum, 91
Phyllosticta spinarum, 163, 180–181 Pythiales, 4
Phylogenetic, 1–13 Pythium, 2, 4
Phylogenomics, 13
Phytolacca dioica, 162, 169, 170 Q
Phytophthora infestans, 237 Quamoclit, 204
Phytophthora ramorum, 52 Quercus variabilis, 161, 174
Phytophthora, 2, 4
Pichia, 7, 69, 110–112 R
P. acaciae, 113, 117, 119
Radulomyces confluens, 212–214
P. angusta, 237
Resupinatus leightonii, 211
P. anomala, 108, 110, 112, 120–123
Rhizoctonia, 11
P. farinosa, 108, 111, 116
R. leguminicola, 206
P. farinosa (sorbitophila), 237
R. oryzae, 81
P. inositovora, 113, 117–119
P. kluyveri, 108, 111 Rhizopus oryzae, 237
P. membranifaciens, 108, 111, 114, 122 Rhizophlyctis, 6
P. stipitis, 237 Rhodotorula, 11
Pinus, 161, 182–183 R. glutinis, 86
Pithomyces Ripartites metrodii, 212
P. chartarum, 276, 278 Ripartites tricholoma, 212
Platycladus orientalis, 163, 180–181 Rosellinia necatrix, 275, 284
Plasmodiophora, 2–5 Rozella, 5, 6
Plasmodiophora brassicae, 3 Rozella allomycis, 5, 6
Plasmopara, 2 Russula lepida, 216–218
Pleosporomycetidae, 9 Russulales, 13
Pleurotus ostreatus, 90 Rust fungi, 10
Pneumocystis, 7
P. nordicum, 354, 355, 357, 359, 361–364, 366, 369, 370 S
Podophyllum emodi, 159 Saccharomyces bayanus, 238
Podospora anserina, 50–52, 69–73, 89 Saccharomyces cariocanus, 47, 53
 Biosystematic Index 399

Saccharomyces castellii, 238 Suillus luteus, 221

Saccharomyces cerevisiae, 41, 42, 46, 47, 50, 52–57, 65, Synechocystis sp., 45, 55
66, 69, 71, 73, 85, 107–116, 118, 119, 121–124, 238, 274,
337, 346, 378, 382 T
Saccharomyces kluyveri, 238 Talaromyces wortmannii, 257
Saccharomyces kudriavzevii, 238 Taphrina, 7
Saccharomyces mikatae, 238 Taphrinomycotina, 7
Saccharomyces paradoxus, 238 Taxomyces andreanae, 156, 198
Saccharomycetales, 84 Taxonomy, 1–13
Saccharomycotina, 7 Taxus, 198
Saitoella, 7 T. brevifolia, 156, 178, 189
Sandoricum koetjape, 159–163 Tectona grandis, 162, 169–171
Sarcodon, 217, 218, 222–223 Thaxteriella pezicula, 280
Saprolegnia, 4 Thelephora, 222–223, 225
Saprolegniales, 4 Thelephorales, 13
Schinus molle, 160, 188, 189 Thraustochytriales, 4
Schizophyllum commune, 81, 82, 238, 384, 386 Tilia Americana, 93
Schizosaccharomyces, 7 Tilletiales, 11
S. japonicus, 238 Tilletiopsis, 11
S. octosporus, 238 Tolypocladium
S. pombe, 46, 238 T. inflatum, 265, 278
Schwanniomyces, 108, 117 T. parasiticum, 279
Sclerosporaceae, 4 T. terricola, 278
Sclerotinia sclerotiorum, 238, 379 T. tundrense, 278
Scytalidium, 280 Torrubiella luteorostrata, 283
Sebacinales, 12, 13 Trametes gibbosa, 94
Selaginella pallescens, 161, 181 Trametes menziesii, 221
Sepedonium, 319 Trechisporales, 13
Septobasidiales, 11 Tremella, 12
Serpula himantoides, 225, 227 T. aurantialba, 223, 226
Sirodesmium, 277 Tremellales, 12
Smut fungi, 11 Tremellomycetes, 12
Sordariaceae, 21, 22 Trichoderma, 86, 92, 93, 383
Sordaria macrospora, 17–36, 89 T. atroviride, 92
Sordariomycetes, 9 T. harzianum, 249
Spartina alterniflora, 94 T. longibrachiatum, 274
Spiromyces aspiralis, 51, 52 T. pseudokoningii, 274
Spizellomyces punctatus, 48, 49 T. reesei, 238, 378, 379, 384
Spodoptera littoralis, 141 T. virens, 238
Sporidiales, 11 T. viride, 279
Sporobolomyces, 11 Trichoderma/Hypocrea, 311
Sporothrix schenckii, 333 Trichoglossum, 8
Spongospora, 4 Tricholoma matsutake, 86, 94
Stachybotrys chartarum, 278 Tricholoma robustum, 85
Stagonospora nodorum, 238 Trichomycetes, 6, 7
Stereum, 220 Trichosporon pullulans, 113, 117
Stereum hirsutum, 224, 226, 275, 278 Tuber borchii, 93
Sterigmatocystin, 133–139, 145 Tuberculina, 11
Straminipila, 4, 5 Tuber melanosporum, 94
Straminipilous fungi, 4 Tulasnella, 12
Streptomyces, 92, 247, 250, 262, 297 Turbina corymbosa, 199–204
S. coelicolor, 346 Tylopilus felleus, 217
S. collinus, 260
S. diastatochromogenes, 264 U
S. thioluteus, 257 Ultrastructure
Strobilurus tenacellus, 249 septum, 9, 11
400 Biosystematic Index

Uncinocarpus reesii, 238 Williopsis, 107–110

Urocystales, 11 W. californica, 108, 110
Uromyces fabae, 93 W. saturnus, 108–110, 120
Uromyces phaseoli, 85 W. saturnus var. mrakii, 108–110, 112, 121
Ustilaginales, 11 Wingea robertsiae, 69
Ustilaginomycetes, 9, 11
Ustilaginomycotina, 9, 11
X
Ustilago cynodontis, 275
Xanthoparmelia scabrosa, 277
Ustilago maydis, 48, 113, 116, 123, 238, 241, 317, 318,
Xylaria, 159–164, 333
379, 385
Xylaria polymorpha, 284
V
Verrucariales, 9 Y
Verticillium, 249, 255 Yeast(s), 7, 11, 12
V. albo-atrum, 238 Yersinia pestis, 339
V. dahliae, 238
V. coccosporum, 281
V. hemipterigenum Z
Zalerion arboricola, 279
Vinca minor, 160, 175
Zoopagomycotina, 6
Vitis vinifera, 122
Zygomycota, 5–7, 13
Zygosaccharomyces bailii, 47, 53, 113, 116, 120
W
Wangiella dermatitidis, 333, 335
Subject Index

A Arbuscular mycorrhizal (AM) fungi, 80, 92

Acetyl transferase, 366 Argadin, 279, 289
Argifin, 280, 289
A ACR-toxin I, 252, 265, 266
Aculeacin A, 279 Armillol, 214
Adaptation, anti-predator, 143 Arthrichitin, 284
Arthrosporone, 214
Adenylation (A) domain(s), 306, 308–311
Ascocarp, 89
Adenylyl cyclase, 32–33
Aflatoxin, 91 (4S) (+)-Ascochin, 184
(S,S)-(+)-Ascodiketone, 184
AFTOL, 2, 5, 8, 10, 12, 13
Ascogenous hyphae, 19
Agroclavine, 204
Alkaloids, ergotamine, 132 Ascogonia, 18, 22, 87
Altechromone A, 188 Ascogonium, 19
Ascospore, 86, 94
Altenusin, 175
Alternaric acid, 175 Asexual development, 139
Alternariol, 175 Aspergillicins, 289
Aspyridones, 301
Altertoxin I, 175
Alveolata, 4 Asteltoxin, 252, 265
Amanitins, 281 ATP citrate lyase, 34
Atpenin(s), 249, 258–261, 267
A mating type, 81
Aureobasidin A, 284, 288
Amino acid(s), 305, 306, 308–315, 317, 322
7-Amino-4-methylcoumarin, 159 Aureothin, 250, 257, 258
Aurovertin B, 265
Ammonia, 85
Aurovertins, 252
AM toxin(s), 290, 316, 320
Amylase, 377, 378 Autoinhibitors, 85
Anastomosis, 80, 81, 88 Autoselection, 119, 123
Autotropism, positive, 80
Angolide, 287
Anhydrofulvic acid, 262
Anidulafungin, 289 B
Antagonistic interactions, 140 Bacterial intein-like sequences (BILs), 44
Antagonists, 146 Basal fungi, 5–7
Antimycin A3a, 247, 249 Basidifferquinone, 92
Antioxidants, 368 Basidiomycetes, ectomycorrhizal, 92, 93
Antisense RNA, 383, 385 Bassianolide, 283, 286
apdR transcription factors, 300 Beauvericins, 282, 286
Apicidin, 278, 288 Beauverolides, 286–288
Apiosporamide, 259 Biomass loss, 142
Apoptosis, 63–74, 86 Biotyping, 123
Aporpinones, 224, 226 b-isopropylmalate dehydrogenase, 26,
Appressorium formation, 234, 238 34, 35
Arborcandins, 280 (+)-1,10-Bislunatin, 166
402 Subject Index

Botryomaman, 185 bacteria, 79, 84, 91–92, 95, 96

Bovistol, 212, 214 cells, 79–81, 83, 85–89, 92–94, 96
Brefeldin A, 182, 183 chemical nature, 79
courtship, 87
C cross-kingdom, 84, 91
extracellular, 80, 81, 83, 89
Calopins, 217
cAMP, 81 functions, 83, 85, 93, 95, 96
Camptothecin, 156, 157, 166, 198 fungus–animal, 93
Capsaicin, 250 fungus–plant, 93
Capsule formation, 240–242 humans, 94
Carbonarone A, 258 individuals, 79–81, 83, 84, 96
Carbon source, 357, 364 insects, 93–95
Carboxypeptidase, 371 intercellular, 79
b-Carotene, 88 interkingdom, 79
Caspofungin, 273, 289 interspecies, 79, 83, 84
Catabolite repression, 357, 358 intra-species, 79, 83, 91
CATs male-female, 89
conidial anastomosis tubes, 81 mammals, 94
C8-compounds, 85 quorum sensing, 84, 91
Cell killing, 111, 112, 118, 120 sexually determined, 83, 88
Cellular death, 81 signalling molecules, 80, 81, 84, 86, 91, 94, 96
Cephalosporin, 320–321 signals, 79, 82, 93, 96
Cercozoa, 4, 5 species, 79–97
Ceriporic acid, 221 vegetative, 80–87, 89
cfs1, 91 Comparative genomics, 378
Chaetocin, 277 Competition, 147
Chaetoglobosins, 178 Complex, 261, 265
Chemo-attractant, 81 Complex I, 247, 249–258, 261–263, 266
Chlamydocins, 281, 288 Complex II, 247, 249, 251, 253–255, 258–262, 266, 267
Chlamydospore, 201 Complex III, 247, 250, 251, 254, 255, 259, 262–263,
2-Chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1, 266
4-dione, 159 Complex IV, 247, 250–252, 254, 263–266
Chloroperox idase, 356 Complex V, 247, 251
Chromalveolata, 4, 5 Complicatic acid, 216
Chromatin structure, 138 Compound 8, 212–214
Chrysogenamide, 171 Condensation (C) domain(s), 306, 308, 313, 315, 322
Chrysotriones, 221, 222 Conditional protein splicing (CPS), 55
Cinnamic acid, 225 Conidia, 81, 85–87, 91, 382
Circadian rhythm, 368 Conidial anastomosis tubes (CATs), 81
Citreoviridin, 252 Conidiation, 86, 89, 91
Citrinin, 355, 356, 361, 364, 370 Conidiogenone, 87
CJ-15,208, 278, 288 Conocenol(s), 211, 212
Clamp cells, 87 Conocenolide, 211, 212
Clavaric acid, 220 Convergent evolution, 362
Clavariopsins, 289 Coprinol, 216
Cleisthothecia, 89 Coprogen(s), 273, 274, 305, 317, 318
Clock-controlled genes, 368 Cortamidine oxide, 224–226
CO2, 85 Creolophins, 216
Cochlioquinone(s), 250, 251 Cross-species microarray hybridizations, 30
Co-evolution, 96 Cucumins, 214
Colletotric acid, 189 Cucurbitacin, 220
Colutellin A, 182 Curtisians, 223
Combinatorial biosynthesis, 323, 324 Cyathin(s), 217
Communication Cyclo(His-Pro), 275
arthropods, 94 Cyclo(leucylprolyl), 214
 Subject Index 403

Cyclo(phenylalanylprolyl), 214 E
Cycloaspeptide A, 173, 174 Echinocandins, 279, 286, 289
Cycloaspeptides, 289 Ecology, 96
Cyclochlorotine, 279, 287, 288 Ecosystem, 96
Cyclopentenons, 175–176 Ectomycorrhizal, 6
Cyclopropane fatty acid synthase, 91 Efflux pump, 366
Cyclosporin A, 311 Efrapeptin D, 265
Cyclosporins, 273, 278, 279, 286, 288, 305, 314–316, 318 Efrapeptins, 265
Cyrmenin B1, 263 EGFP, 25, 28, 29
Cyrmenins, 263 Eleganthol, 217
Cytochrome c, 64–66 Elicitors, 92, 93
Cytochrome P450, 91 eln2, 91
Cytosporone D, 170 eln3, 91
Elongation/extending modules, 307
D Emodepsin, 289
Emodin-1,6-dimethylether, 174
Dacrymenone, 216, 217
Endomycorrhizae, 92
Daldinone C, 188
Endonuclease, 41–44, 46, 48–54, 57
Daldinone D, 188
Enniatins, 282, 286, 287, 290
-Decalactone, 252, 253
Entomopathogenic, 141
Decomposer communities, 141
Environment factors, 135
Defence chemicals, 96
Epialtenuene, 175
5-Demethylovalicin, 211, 212
Epibiotic fungus, 201–203
3,4-Deoxy-3,4-didehydromultiplolide A, 166
Epibiotic niche, 199, 201
Deoxyfunicone, 257, 258
Epichlicin, 181, 182
5-Desoxyilludosin, 212, 213
Epicorazines, 278
Destruxins, 284, 286, 287, 290
(+)-Epicytoskyrin, 166
Development
Epimerization domains, 313–315
fruiting body, 88–91, 93
6-Epitaedolidol, 178
sexual, 83, 87, 89
Equisetin, 300
Dicerandrols, 170
Eremoxylarin, 163
Dichomitol, 214, 215
Ergoline alkaloid, 197–206
Dichomitone, 214, 215
Ergoline alkaloid biosynthesis, 198, 199, 201, 202, 204
Differential expression, 364
Ergopeptides, 273, 290
5,6-Dihydro-5,6-epoxymultiplolide A, 166
Ergosterol, 219
2,3-Dihydro-5-methoxy-2-methylchomen-4-one, 183
Ergostra-4,6,8(14),22-tetraen-3-one, 219, 220
3,12-Dihydroxycadalene, 166
Ergot alkaloids, 157, 319, 321, 322
3,12-Dihydroxycalamenene, 166
Ergovaline, 276
6,9-Dihydroxy-3(15)-caryophyllen-4,8-dione, 214, 215
EST sequencing, 238
2b,13-Dihydroxyledol, 214, 215
Eukaryotic toxins, 120
Dikarya, 7
Evolution, 96
Dimargaritales, 7
Excavata, 2
Dimerumic acid, 274
Export, 361
2,7-Dimethoxy-6-(1-acetoxyethyl) juglon, 189
Expression analysis, 357, 360, 362
Dimethylallyltryprophan (DMAT), 322
Expression optima, 365
4-[g,g-Dimethylallyl]tryptophan synthase, 202
Expression pattern, 364, 365
Dimorphism, 86–87
Extein
Dioxapyrrolomycin, 247, 248
C-extein, 41, 43–45, 56
Dispensable (CD) chromosome, 320
N-extein, 41, 43–45
DNA binding factor, 357
DNA-mediated transformation, 24, 26–27
Dominant marker, 26 F
Drimene-2,11-diol, 212, 214 False truffles, 94
Drug development, 240 1,6-Farnesadiene-3,10,11-triol, 211
DsRed, 28 Farnesol, 69, 85, 86, 91, 241
dsRNA, 107, 112–116, 119, 121 Fatty acids, 86
404 Subject Index

fatty acid biosynthesis, 333 tip-to-side, 82

fatty acid synthase (FAS), 133, 333 tip-to-tip, 82
Favolon(s), 219 vegetative, 81–84
Fericrocin, 274
Ferrichrome, 274, 275, 305, 309, G
317–319
Gametangia, 88
Ferrichrysin, 274
Gametes, 88
Ferricrocin, 274
Gene
Fertilization, 87, 89
cluster(s), 316, 319–324
Fitness parameters, 140
expression, 357, 359, 364, 367, 368
FKI-1033, 283
mining, 378
Flagellum, 4, 6
targeting, 383–386
Flavenoids, 92
transcription, 363
Flavipucine, 259
Genetic engineering, 377, 381, 382
Fluorescence microscopy, 27–30
Genome mining, 297–302, 340
Fluorescent dyes, 27, 29
Genome sequencing, 378, 379
Fomitellic acid, 219
Genomic(s), 146, 147
Food choice, 143
Geosmin, 211
Fostriecin, 253
1(10),4-Germacradiene-2,6,12-triol, 211
FR190293, 279
Germination, 84–86, 92–94, 239
FR227673, 279
Gerronemins, 222, 223
FR235222, 282, 288
Glandular cell, 201, 202
FR901379, 273, 279
Gliotoxin, 67, 273, 277, 287, 288
FR901469, 281, 284, 287
Gliotoxin gene cluster, gliZ, 135
Fruiting body
Gliovictin, 172, 273, 277, 287, 288
aroma, 94, 95
Gloeophyllols, 214
ascomycetes, 89
Gloeophyllone, 212, 214
basidiomycete, 88, 90
Glomosporin, 285, 289
development, 17–19, 21, 22, 27, 30, 34
a-(1,3)-Glucan, 85
induction, 89, 92, 93
Glutamate dehydrogenase, 239
primordium, 89
Glycosylation, 377
stipe elongation, 91
Glycosyltransferase, 91
Fumitremorgins, 276, 288
Golmaenone, 275, 276
Fumonisin B1, 255
G proteins, 31–33
Functional genomics, 383
Green fluorescent protein (GFP), 55
Fungicide(s), 203, 363, 366, 371
Growth
amphotericin B, 240
density-dependent, 84, 85
azole, 234, 240, 241
pseudohyphal, 86
strobilurin, 234
Growth phase, 357, 363, 373
voriconazole, 241
Fungivore(s), 142, 147
Fungivory, 139, 141 H
foraging strategies, 142 Hammerhead ribozymes, 383, 385
Fungus/plant symbiotum, 204 Hartig net, 89, 93
Funicone, 257, 258 Harzianopyridone, 258, 261
Funiculosin, 259, 262, 263 HC-toxin(s), 280, 288, 290, 310, 315, 316, 319
Fusarin, 300 Hedgehog, 43, 44, 46
Fusaristatins, 181 Helminth, 247–267
Fusigen, 274 Helminth complex I, 251–258
Fusion Helminth complex II, 261–267
frequencies, 81, 82 Helper bacteria, 92
hyphal, 80–83, 87 2-n-Heptyl-4-hydroxyquinoline N-oxide, 262
mating type, 83, 87 Heterokaryon incompatibility, 81
peg-to-peg, 82 Heterokaryosis, 81
tip-to-peg, 82 Heterologous expression, 299, 345–347
 Subject Index 405

Heterothallic, 18–22, 31, 36, 87, 89, 94 GLT intein, 11

Himanimides, 225, 227 inhibitory agents, 56
Himeic acid A, 257, 258 large intein(s), 43, 46, 47, 50, 51, 53
1H-indole-3-carboxylic acid, 226, 227 mini-intein, 43, 46, 47, 49–51
Histone deacetylase (HDAC), 138 PRP8 intein, 46–50
HO gene, 41, 52, 54 split intein, 45, 55, 56
Homing, 42–44, 46, 51–54 structures, 43
attraction, 81 VMA1 inteins, 46, 49, 52–54
hyphal, 80 Internal transcribed spacer (ITS), 202, 203
interspecies, 83 Introns, 41, 42, 49, 51–53, 316–318, 320, 322
spores, 81, 83 Irofulven, 212, 213
Homologous recombination (HR), 22, 27, 299, 383 Irpexans, 218
Homothallic, 18–22, 31, 89 Isariins, 286, 287
Horizontal gene transfer, 342–344 Isocochlioquinone A, 250, 251
Hormonemate, 182–183 Isopestacin, 178
Hormones, 86 Isovaleric acid, 85
Host specificity, 199, 205 Iterative NRPSs, 309, 317
HUN-7293, 288
195-A, 257, 259 J
Hybrid product(s), 298
JM47, 280, 282, 288
Hydroperoxy fatty acids, 86
Hydroxamic acid, 85
K
3-Hydroxy-1-(2,6-dihydroxyphenyl)-butan-1-one, 183
3b-Hydroxylanosta-8,24-dien-21-oic acid, 219 K1, 109, 111, 112, 114–116, 120–122
Hydroxymellein, 159, 166, 184, 185 K28, 112, 114–116, 120, 124
7-Hydroxy-8-methoxy-3,6-dimethyldi-benzofuran-1, Kazusamycin A, 253
4-dione, 159 3-Ketotauranin, 181
1-(2-Hydroxy-6-methoxyphenyl) butan-1-one, 183 Killer, 107–124
(+)-(5S,10S)-40-Hydroxymethyl-cyclozonarone, 181 Killer toxin-like antibodies, 121
(+)-10a-Hydroxy-4-muurolen-3-one, 219 Kinase, 368
Hydroxyquinoline N-oxide (HQNO), 262 Kirromycin, 260
1-Hydroxy-3-sterpurene, 212, 214 Knockout, 340, 345–346
3a-Hydroxytauranin, 181 ku70, 25, 27
12-Hydroxytauranin, 181
Hygrophorones, 221, 222 L
Hygromycin B, 26 b-Lactam, 320, 321
Hypericin, 158 LaeA, 324
Hyphal fusion, 29, 34–36 L-alanyl-L-tryptophan anhydride, 275, 276
Hyphal interference, 83 Laschiatrion, 219
Leaianafulven, 213
I Lepidamine, 216, 217
ich1, 91 Lepidolide, 217, 218
Illinitones, 217 Leptomycin B, 252, 253
Illudin, 212–214 Leptosins, 288
Immunity phenotype, 120 Leptosphaeric acid, 187
Immunofluorescence, 28, 29 Leucinostatin A, 266
Importin a, 370 Leucopaxillone, 219, 220
Indolizidine alkaloid, 206 Lifespan, 71–74
Induction, 363, 364, 366–368, 372 Light, 363, 368–371
Inhibitor, competitive, 365 Lignin, 380
Initiation/starting module, 307 Limacellone, 216, 217
Insect feeding deterrent, 319 Linear dsDNA plasmids, 116
Insect mortality, 145 Linear NRPS, 308, 309
Intein(s) Lipoxygenases, 86
activity, 49–52 LL15G256g, 281, 284
evolution, 53–54, 57 Llicicolin H, 259, 262, 263
406 Subject Index

Locoism, 206 Mycotoxin(s), 140, 147, 157–158, 378

LpsA, 322 Mycotoxin patulin, 91
Lumichrome, 174 Myxobacteria, 250, 263
Myxothiazol, 263
Myxothiazol A, 263
M
Mactanamide, 275, 276
Malformins, 279, 288, 289
N
Massarilactones, 189 N-acyl homoserine lactones, 91
Massively parallel signature sequencing (MPSS), NADH-fumarate reductase (NFRD), 247, 249, 251–258,
241–242 261, 266
Mating Nafuredin, 251–254, 257, 265, 266
A mating type, 81 Nafuredin-g, 254, 255
basidiomycetes, 81, 87 Natural product(s), 297, 298, 301, 302, 331, 341
bipolar species, 81, 87 Necrosis, 135
B mating types, 81, 82 Neoaureothin, 257, 258
(+) mating type, 88 Neocallimastigomycota, 6
(
) mating type, 88 Neoechinulin, 275, 276, 288
mating type loci, 81, 87 Neofrapeptins, 289
mating type locus, 81, 82, 87 Nitidon, 221
switching, 52, 54 Nitrogen, 36, 363
tetrapolar species, 81, 87 Nitrogen source, 357, 359
transcription factors, 87 N-methylation domain, 314
type, 18, 20–22, 31, 33 Nodulisporin A, 183
Maytenine, 226 Non-linear NRPS(s), 308, 309
Melanin, 234 Non-ribosomal peptides (NRPs), 305, 313, 335,
Mellein, 166, 167, 169, 172, 185 339–341, 347
Messenger molecules, 84, 96 cephalosporin, 132
Metabolic profile, 298 cyclosporine, 132, 133
Metabolomics, 235, 380, 381 gliotoxin, 132
Metacaspase(s), 65, 66, 70, 72–74 b -lactam antibiotics, 133
Metagenomics, 378 penicillin, 132
14-Methoxytajixanthone-25-acetate, 175 Non-ribosomal peptide synthetases (NRPSs), 132, 133,
Methyl salicylic acid synthase, 361 298, 305, 324
Methyltransferase, 138, 356 Nourseothricin, 26
Mevinic acid, 166 Nucleus, 368, 370
Micafungin, 273, 289
Microarray(s), 30, 146, 233, 235–242, 360, 364, O
366, 380
Microsporidia, 5, 6 8-O-acetyl-5,6-dihydro-5,6-epoxymultiplolide A,
Militarinone, 301 166
Militarinone D, 259 8-O-acetylmultiplolide A, 166
Mitochondria, 63–66, 70 Occhratoxin A, biosynthesis, 353–373
Mode of action, 233–235, 240, 241 3-Octanol, 86, 95
Module(s), 306–309, 311–315, 319, 322, 324 3-Octanone, 86, 94
Molecular clock, 368, 370 1-Octen-3-ol, 85, 86, 91, 93–95
Montadial, 224, 226 Oligomycin A, 264
Morphogens, 89 O-methyltransferase, 91
Mucidin, 263 Omics, 379–381, 386
Multiplolide A, 164, 166 Omphadiol, 213
Mulundocandins, 279 Omphalotins, 281, 282, 286, 287, 289
Mutagenesis, 22, 26 Ostreolysin, 90
Mycelianamide, 276 Oudemansin, 222
Mycocines, 107 Oudemansin A, 263
Mycophagy, 94 13-Oxo-9(Z),11(E)-octadecadienoic acid, 212
Mycorrhiza, 7, 13 Oxylipins, 1-octen-3-ol, 86
 Subject Index 407

P Phomaxanthones A, 170
Pachydermin, 225, 227 Phomol, 167, 168
Paclitaxel, 198 Phomopsidone, 169
Paecilaminol, 249, 254–255 Phomopyronol, 167
Palmarin, 252, 253 40 -Phosphopantetheine transferas, 311–313
Parasitism, 147 Photocleavage, 371
Partially reducing PKS, 337 Phyllospinarone, 181
PaT, 119, 120 Phylogenetic tree, 202
Pathogenic, 141 Phylogeny, 6, 342–344
Pathogenicity, 133 Physicion, 174
Pathogens, 80, 92 Piericidin A, 247, 249, 250
Pathway regulation, 347 Pigment(s)
Pathway-specific regulatory genes, transcription bikaverin, 333, 347
factors, 299 melanin, 331, 333, 342, 345
Paxilline, 186 Pironetin, 252, 253
Peltate glandular trichome, 200, 201 PKS-NRPS hybrid, 300
Penicidones, 174 Plant–fungus symbioses, 202
Penicillenols, 170 Pleofungins, 288
Penicillin, 305, 311, 312, 316, 318, 320–321, PNa, 362
324, 377 Pneumocandin, 273, 279, 281
Penicillin G, 276 Podophyllotoxin, 158–159
Penicillone, 186 Polyketide(s), 355–357, 360–366, 371
Peniprequinolone, 172 aflatoxin B1, 132
Penochalasin A, 178 aflatoxins, 133
Peptaibols, 311, 313, 315 classification, 334
Peptides highly reducing PKS, 337–339
pheromone-like, 83, 84 lovastatin, 132, 133
QSP1, 84, 85 non-reducing PKS, 335–337, 342
synthetase, 357, 361 polyketide biosynthesis, 333–334
Peptidyl carrier protein (PCP) domain, 306, 311 polyketide synthase (PKS), 334–341
Per5, 23, 34, 35
post-PKS modification, 341–342
Percyquinnin, 220
Polyketide synthase(s), 133, 297
Perithecia, 89, 91
citrinin, 356, 361, 364
Pestafolide A, 179
40 Ppant cofactor, 312
Pestahivin, 284, 288
Predation, 147
Pestalotiopsolide A, 178
Preservatives, 363, 366, 367, 371
Pestaphthalides, 179
Primin, 185
Petasol, 216, 217
pro1, 22, 30, 34, 35
Petriellin A, 284, 287, 289
pro4, 34, 35
PF1022A, 273, 284, 288
pro11, 30, 34
PH, 357, 359, 360, 363, 364, 366, 371
pro22, 30, 34, 35
Phaeosphaeride(s), 183
ppro40, 28, 34, 35
Phenalamide A2, 258, 259
pro41, 28, 30, 34, 35
Phenoxan, 250
Promoter(s), 357–360, 369
Phenylalanine, 355–357, 365, 371
Promoter exchange, 299, 300
Pheromones, 31, 32, 66, 69, 70
Protein–protein interactions, 46, 54, 55
autocrine functions, 83
Protein purification, 45, 54, 56
lipo-peptide, 84
paracrine functions, 83 Protein splicing, 41–46, 50, 55–57
peptide, 83, 84, 96 Proteome profiling, 242
pheromone-like peptides, 83, 84 Proteomics, 235, 243, 380
receptor(s), 31, 32 Protists, 2, 4
non-mating-type, 83 Prp8 gene, 46, 49, 57
PR toxin, 216, 217
seven-transmembrane Gprotein-coupled
Prugosene A1, 257, 259
type, 83, 87
408 Subject Index

Psathyrellon, 212, 214 RNA polymerases, 46, 51

Pseudodestruxins, 283 RNA-silencing, 341, 346
Pseudo-flowers, 93 Roquefortins, 288
Pseurotin A, 173 Rotenone, 247, 249, 250
psi, 86, 89 RT-PCR, 146
psiAa, 86
psiBa, 86 S
psiCa, 86 S-Adenosyl-methionine (SAM), 314
Psychrophilins, 280, 289 Sambutoxin, 259, 262, 263
Pteratides, 282 Sansalvamide A, 284, 289
Pterulinic acid, 250, 251 Saprophytic, 141
Pterulone, 250, 251 Saprotrophs, 85, 93
Pyrenocines, 186 Sarcodonins, 217
Scabronines, 217
Q Scabrosin, 277, 278
Quorum sensing Sclerotia, 86
cell-density, 84 Sclerotium, 199
cross-kingdom, 84, 91 Scytalidamides, 274, 280
inhibitor, 91 Secondary metabolism, 297–299
population response, 84 Secondary metabolite biosynthesis, 298
Quorum sensing inhibitor (QSI), 91 Secondary metabolite cluster
aft A-Y, 137
silent, 139
R
Secondary metabolite clusters
Racemase(s), 314–315 aflatoxin/sterigmatocystin gene clusters, 133
Radudiol, 213 aflR, 133
Radulactone, 213 Secondary metabolites, 142, 305, 323
Radulol, 213 aflatoxin B1, 132, 141
Radulones, 213, 214 aflatoxins, 132, 133, 135
Rapamycin, 55 cephalosporin, 132
Receptor cyclosporine, 132, 133
blue light, 370 epipolythiodioxopiperazines (ETP), 135, 138
red light, 371 fumonisins, 132
Reductase domain, 315 gibberellin GA3, 132
Regulation gliotoxin, 132, 135, 138, 141
epigenetic, 138 lovastatin, 132, 135
global, 135 penicillin, 132, 135
LaeA, 139 regulator genes, 146
pathway-specific, 135 sterigmatocystin, 135, 145
VeA, 138 Secreted factor, 82
VelB, 138 Secretion, 377
Regulator Secretory glands, 201, 203, 205
LaeA, 135, 138, 142 Seed-transmitted fungus, 205
Regulatory gene(s), 359, 360, 364 Self-inhibitors, 85
Remote sensing, 80 Self-protection mechanism, 366
Reporter strain, 366 Sensors, light, 370
Reproduction Septa, 9, 12
asexual, 86–87 Serial analysis of gene expression (SAGE), 233,
sexual, 87, 88 241–242
Resistance, 233, 234, 240–241 Serialynic acid, 224, 226
Responsive elements, 369 Serine protease, 238
Restriction enzyme-mediated Serinocyclin A, 280, 289
integration (REMI), 346 Sesquiterpene(s), 85, 88, 93, 94, 96, 204
Rhodotorulic acid, 274, 275 Sevastelins, 288
Riparols, 211 Seven-trans-membrane G protein-coupled receptors,
RNA interference (RNAi), 383, 385, 386 83, 87
 Subject Index 409

Shamixanthone, 175 Substrate, 355, 357, 363, 364

Shearamide A, 290 Suillumide, 221
Shimalactone A, 257, 259 Surface-active compounds, 90
Siccanin, 247, 249, 261, 262 Symbiotum, 198, 204, 206
Siderophores, 85, 305, 317–319 Synnemata, 201
Signals System biology, 380
attraction, 93 Systemic infection, 199
autoregulatory, 84
bioelectrochemical, 79 T
Ca2+-mediated, 92 Taedolidol, 178
chemical, 81, 92 Taijixanthone, 175
electrical fields, 79 Talaroflavone, 175
extracellular, 80, 81 Tauranin, 181
1-octen-3-ol, 93 Taxol, 156–157, 189
repellence, 93 Taxonomy, 1, 2, 5, 6, 11, 13
volatile organic compounds (VOCs), 86, 91 Teaching, 1, 2, 6, 13
Signal transduction, 368 Teliospores(s), 11
heterotrimeric G protein, 138 Temperature(s), 355, 360, 363, 364, 366,
protein kinase A, 138, 139 367, 371–373
Silage, 122 Tenellin, 301
Silent gene cluster(s), 324 Tentoxin, 265
Sirenin, 88 Tenuazonic acid, 175
Sirodesmin PL, 135 Terpenes, 87, 132, 133, 201
Slime moulds Gibberellin GA3, 132
acrasids, 2 Terphenyls, 222, 225
dictyostelid, 3 Terrestin(s), 223
myxogastrid, 3 Tetrad analysis, 25–26, 36
protostelid, 3 Thallus, 4, 6
Soil fauna, 141 Thelephantins, 223
Species interaction, 139 Thelephoric acid, 225
Specificity conferring code, 310 Thioesterase domain, 315
Sporangiospores, 6 Thiolation (T), 308
Spores Thiolation domains, 311–313
germination, 85, 92 Thiotemplate mechanism, 306
meiotic, 83, 90 Tintinnadiol, 218
mitotic, 83 a-Tomatine, 68
Sporidesmins, 276, 288 Toxin(s)
18SrDNA, 202 aflatoxin, 332, 335, 341, 342, 344, 345
Stemphone A, 251 citrinin, 331, 335
Sterigmatocystin, 91 compactin, 333, 334, 338, 345
Sterigmatocystin clusters cytochalasin, 331, 333, 340
and aflatoxin clusters, 135 fumonisin, 338, 342
aflJ, 135 lovastatin, 331, 338, 342
aflR, 135 squalestatin, 338
Sterins, 224, 226 sterigmatocystin, 336, 345
Sterol biosynthesis, 234, 235 T-toxin, 331, 333, 338, 342, 346
Storage fungi, 355 zearalenone, 335
Stress Trametenamides, 221
adaptation, 363, 366, 368, 371 Transcriptional level, 364
oxidative, 367, 368 Transcriptional regulation, 369
responses, 368, 373 Transcription factor(s), 33–34, 87, 135, 299–301
Striatin(s), 30, 34, 217 Transcriptomics, 380, 381
Strigolactones, 92 Transformation
Strobilurin A, 247, 248, 251, 263 Agrobacterium tumefaciens mediated, 382
Strobilurins, 210, 218, 219, 224 electroporation, 382
Subcuticular oil storage cavity, 201, 202 protoplasts, 382
410 Subject Index

Trapoxins, 280 VMA1 gene, 41, 46, 52, 54, 57

Tremellin, 223, 226 Volatile oil, 201, 203
Triacetylfusigen, 274, 275 Volatile organic compounds (VOCs), 85, 86, 89,
Trichogynes, 87 91–96
3,11,12-Trihydroxycadalene, 166
3,9,12-Trihydroxycalamenenes, 166
(4E)-6,7,9-Trihydroxydec-4-enoic, 166 W
(4E,6E)-2,4,6-Trimethyloacta-4,6-dien-3-one, 183
2,3,5-Trimethyl-6-(3-oxobutan-2-yl)-4H-pyran-4-one, Water activity, 357, 363, 366, 367
257, 258 WD40 repeat protein, 34, 35
Trisporic acid, 88 WF11899, 279, 281
TRNA-cleaving toxins, 124 WF-16775, 258, 260
TRNase, 118–120 White collar1, 368
T-RNA synthetase White collar2, 368
phenylalanine, 355–357, 365, 371 Wine, 110, 112, 121–123
Truffles, 93, 94 Woronin body, 28, 34
Tyrosol, 84 WWdomain protein, 34

U X

Ukulactones, 249, 257–259 Xyloallenolide B, 163

Uncoupler, 252, 265–266 Xyloester A, 163
Uncoupling, 265, 266 Xyloketal J, 163
Unguisins, 280
UV-mutagenesis, 346 Y
Yeast, 65, 66, 69–73
V Yeast cells, 65, 70, 72
Vegetative incompatibility, 81 Yoghurt, 122
Velvet A, 369
Vermistatin, 257, 258 Z
Verruculogens, 276, 288
Verticilide, 285, 289 Zinc cluster, 34
Verticipyrone, 255–257, 261, 266 Zinc finger transcription factor, 34
Vertihemiptellide(s), 277, 278, 288 Zoospore(s), 3–6
Vibralactones, 220 Zygomycetous, 6
Virulence, 331, 333, 339, 342 Zygospore(s), 6, 88
Virulence factor(s), 319 Zygotropism, 88
VM 3298-2, 216, 217 Zymocines, 107, 112, 117–120, 122